Binding members for interleukin-4 receptor alpha (IL-4Ra) - 173

ABSTRACT

Binding members, especially antibody molecules, for interleukin (IL)-4 receptor alpha (IL-4Rα), and their therapeutic use e.g. in treating or preventing disorders associated with IL-4Rα, IL-4 and/or IL-13, examples of which are asthma and COPD.

This application is entitled to priority pursuant to U.S.C. 35 §119(e)to U.S. Provisional Application Ser. No. 61/015,869, filed Dec. 21,2007, which is incorporated herein by reference in its entirety.

This invention relates to binding members for interleukin (IL)-4receptor alpha (IL-4Rα, also referred to as CD124), especially antibodymolecules, and their therapeutic use e.g. in treating or preventingdisorders associated with IL-4Rα, IL-4 and/or IL-13, examples of whichare asthma and COPD.

The human IL-4Rα subunit (Swiss Prot accession number P24394) is a 140kDa type 1 membrane protein that binds human IL-4 with a high affinity(Andrews et al J. Biol. Chem. (2002) 277:46073-46078). The IL-4/IL-4Rαcomplex can dimerize with either the common gamma chain (yc, CD132) orthe IL-13Ralpha1 (IL-13Rα1) subunit, via domains on IL-4, to create twodifferent signalling complexes, commonly referred to as Type I and TypeII receptors, respectively. Alternatively, IL-13 can bind IL-13Rα1 toform an IL-13/IL-13Rα1 complex that recruits the IL-4Rα subunit to forma Type II receptor complex. Thus, IL-4Rα mediates the biologicalactivities of both IL-4 and IL-13 (reviewed by Gessner et al,Immunobiology, 201:285, 2000). In vitro studies have shown that IL-4 andIL-13 activate effector functions in a number of cell types, for examplein T cells, B cells, eosinophils, mast cells, basophils, airway smoothmuscle cells, respiratory epithelial cells, lung fibroblasts, andendothelial cells (reviewed by Steinke et al, Resp Res, 2:66, 2001, andby Willis-Karp, Immunol Rev, 202:175, 2004).

IL-4Rα is expressed in low numbers (100-5000 molecules/cell) on avariety of cell types (Lowenthal et al, J Immunol, 140:456, 1988), e.g.peripheral blood T cells, monocytes, airway epithelial cells, B cellsand lung fibroblasts. The type I receptor predominates in hematopoieticcells, whereas the type II receptor is expressed on both hematopoieticcells and non-hematopoietic cells.

IL-4Rα polymorphisms in the human population have been described(reviewed by Gessner et al, Immunobiology, 201:285, 2000) andassociation with IgE levels or clinical atopy has been reported in somepopulations. For instance, V75R576 IL-4Rα is associated with allergicasthma and enhanced IL-4Rα function (Risma et al. J. Immunol.169(3):1604-1610, 2002).

Several lines of evidence support an important role for the IL-4/IL-13pathway in asthma pathology (reviewed by Chatila, Trends in MolecularMed, 10:493, 2004), and also in a range of other conditions, as listedelsewhere herein. Increased secretion of IL-4 and IL-13 are believed toboth initiate and maintain the disease process. IL-13 is thought to bethe dominant partner in triggering airway hyperresponsiveness (AHR),mucus hypersecretion and airway remodelling, whereas IL-4 is has beensuggested to be the main inducer of Th2 polarisation and IgE production(Wynn, Annu Rev Immunol, 21: 425, 2003).

The role of IL-4Rα in asthma is further supported by evidence fromanimal models of disease. Administration of a functional murine IL-4Rantagonist (IL-4 mutant; C118 deletion) during allergen challenge withovalbumin (OVA, a model allergen) inhibited the development of allergicairway eosinophilia and AHR in mice previously sensitized with OVA(Tomkinson et al. J. Immunol. 166(9):5792-5800, 2001). Furthermore, anumber of in vivo studies have demonstrated positive effects of blockingeither IL-13 or IL-4 in animal models of asthma. For instance,therapeutic dosing with an anti-IL-13 mAb in a chronic model ofOVA-induced persistent airway inflammation inhibited AHR, haltedprogression of subepithelial fibrosis and inflammation and restoredmucus hyperplasia to basal levels (Yang et al., J. Pharmacol. Exp. Ther.313(1):8-15, 2005). In a mouse OVA model, inhibition of IL-4 by ananti-IL-4 antibody showed a marked reduction in eosinophil infiltrationwhen administered during immunization (Coyle et al., Am J Resp Cell MolBiol, 13:54, 1995). In a similar model, IL-4-deficient mice hadsubstantially fewer eosinophils in bronchoalveolar lavage and much lessperibronchial inflammation after OVA challenge (Brusselle et al., ClinExp Allergy, 24:73, 1994).

In humans, phase IIa studies showed that an IL-4Rα antagonist (aso-called IL-4 mutein) reduced an allergen-induced late asthmaticresponse and attenuated the resting inflammatory status of the lungs inasthma patients (Wenzel et al., Lancet, 370:1422, 2007). These humandata further strengthen the notion that an IL-4Rα antagonist may provideclinical utility in asthma.

In addition to its role in asthma, IL-4Rα has been linked with a numberof other pathologies, e.g. as follows:

Chronic Obstructive Pulmonary Disease (COPD) includes patientpopulations with varying degrees of chronic bronchitis, small airwaydisease and emphysema and is characterised by progressive irreversiblelung function decline that responds poorly to current asthma basedtherapy. The underlying causes of COPD remain poorly understood. The“Dutch hypothesis” proposes that there is a common susceptibility toCOPD and asthma and therefore, that similar mechanisms may contribute tothe pathogenesis of both disorders (Sluiter et al., Eur Respir J, 4(4):p. 479-89, 1991). Zheng et al (J Clin Invest, 106(9):1081-93, 2000) havedemonstrated that overexpression of IL-13 in the mouse lung causedemphysema, elevated mucus production and inflammation, reflectingaspects of human COPD. Furthermore, AHR, an IL-13 dependent response inmurine models of allergic inflammation, has been shown to be predictiveof lung function decline in smokers (Tashkin et al., Am J Respir Crit.Care Med, 153(6 Pt 1):1802-11, 1996). A link has also been establishedbetween an IL-13 promoter polymorphism and susceptibility to developCOPD (Van Der Pouw Kraan et al., Genes Immun, 3(7): 436-9, 2002). Thesigns are therefore that IL-4/IL-13 pathway, and in particular IL-13,plays an important role in the pathogenesis of COPD.

IL-13 may play a role in the pathogenesis of inflammatory bowel disease.Heller et al. (Heller et al., Immunity, 17(5):629-38, 2002) report thatneutralisation of IL-13 by administration of soluble IL-13R.alpha.2ameliorated colonic inflammation in a murine model of human ulcerativecolitis. Correspondingly, IL-13 expression was higher in rectal biopsyspecimens from ulcerative colitis patients when compared to controls(Inoue et al., Am J Gastroenterol, 94(9):2441-6, 1999).

In addition to asthma, the IL-4/11-13 pathway has been linked to otherfibrotic conditions, like systemic sclerosis (Hasegawa et al., JRheumatol, 24(2):328-32, 1997), pulmonary fibrosis (Hancock et al., Am JRespir Cell Mol Biol, 18(1): 60-5, 1998), parasite-induced liverfibrosis (Fallon et al., J Immunol, 164(5): 2585-91, 2000; Chiaramonteet al., J Clin Invest, 104(6): 777-85, 1999; Chiaramonte Hepatology34(2):273-82, 2001), and cystic fibrosis (Hauber et al., J. Cyst Fibr,2:189, 2003).

IL-4 and to some extent IL-13, are crucial for B cell mediatedactivities, such as B cell proliferation, immunoglobulin secretion, andexpression of FcepsilonR. Clinical applications of an IL-4Rα inhibitorinclude for example, use in allergy therapy to suppress IgE synthesis(including for example atopic dermatitis and food allergy), use intransplation therapy to prevent transplant rejection, as well assuppression of delayed-type hypersensitivity or contact hypersensitivityreactions.

Il-4R antagonists may also find use as adjuvants to allergyimmunotherapy and as vaccine adjuvants.

Antibodies to IL-4Rα have been described. Two examples are theneutralizing murine anti-IL-4Rα monoclonal antibodies MAB230 (clone25463) and 16146 (clone 25463.11) which are supplied by R&D Systems(Minneapolis, Minn.) and Sigma (St Louis, Mo.), respectively. Theseantibodies are of the IgG2a subtype and were developed from mousehybridomas developed from mice immunised with purified recombinant humanIL-4Rα (baculovirus-derived). Two further neutralizing murincanti-IL-4Rα antibodies M57 and X2/45-12 are supplied by BD Biosciences(Franklin Lakes, N.J.) and eBioscience (San Diego, Calif.),respectively. These are IgG1 antibodies and are also produced by mousehybridomas developed from mice immunized with recombinant solubleIL-4Rα.

Fully human antibodies are likely to be of better clinical utility thanmurine or chimeric antibodies. This is because human anti-mouseantibodies (HAMA) directed against the FC part of the mouseimmunoglobulin are often produced, resulting in rapid clearance andpossible anaphylactic reaction (Brochier et al., Int. J. Immunopharm.,17:41-48, 1995). Although chimeric antibodies (mouse variable regionsand human constant regions) are less immunogenic than murine mAbs, humananti-Chimeric antibody (HACA) responses have been reported (Bell andKamm, Aliment. Pharmacol. Ther., 14:501-514, 2000).

WO 01/92340 (Immunex) describes human monoclonal antibodies against IL-4receptor generated by procedures involving immunization of transgenicmice with soluble IL-4R peptide and the creation of hybridoma cell linesthat secrete antibodies to IL-4R, the principal antibody 12B5 isdisclosed as being an IgG1 antibody and fully human. WO 05/047331(Immunex) discloses further antibodies derived from 12B5 (renamed H1L1)via oligonucleotide mutagenesis of the VH region. Each mutated VH chainwas paired with one of 6 distinct VL chains to create a small repertoireof antibody molecules.

WO 07/082,068 (Aerovance) discloses a method of treating asthmacomprising administering a mutant human IL-4 protein havingsubstitutions of R121D and Y124D. The specification teaches that suchIL4 mutein administered in a pharmaceutical composition can antagonisethe binding of wild type huIL-4 and wild type huIL-13 to receptors.

WO 08/054,606 (Regeneron) discloses particular antibodies against humanIL-4R that were raised in transgenic mice capable of producing humanantibodies.

There are advantages and benefits in the discovery and development of anantibody to human IL-4Rα that also exhibits cross-reactivity to theorthologous protein from another species, for example cynomolgus monkey.Such an antibody would facilitate the characterization of suchantibodies with respect to pharmacology and safety in vivo. Potency oraffinity to another species, which is for example less than 10-folddifferent than the human activity may be appropriate for such anevaluation. However, the human IL-4Rα protein displays a relativelylittle similarity to the orthologous IL-4Rα protein from other speciesexcept chimpanzee. Therefore, the discovery of high affinity and potencyantibodies appropriate for clinical use with cross-reactivity to aspecies widely considered suitable for safety and toxicologicalevaluation for clinical development would be very challenging.

Through appropriately designed selection techniques and assays, theinventors have developed binding members for IL-4Rα that inhibit thebiological activity of human and cynomolgus monkey IL-4Rα.

As detailed in the Examples, from an initial lead identification programthe inventors selected a single antibody molecule to human IL-4Rα thatalso exhibited some, but weak, binding to and functional neutralisationof cynomolgus IL-4Rα. Following a planned and defined process oftargeted and random mutagenesis and further selection of mutants fromthis parent antibody molecule, a larger panel of antibody molecules withgreatly improved properties was developed. VH and VL regions, includingthe complementarity determining regions (CDRs) of the parent antibody(Antibody 1), and of the optimised antibodies, are shown in FIGS. 1, 2,3 and 4. These antibody molecules, VH, VL, CDRs, and binding memberscomprising one or more of the CDRs, form aspects of the presentinvention. In addition to wild-type IL-4Rα the binding members of thepresent invention have also been found to bind 175V IL-4Rα, a commonhuman variant.

Described herein are binding members that neutralise the biologicaleffects of IL-4Rα with high potency, bind IL-4Rα with high affinity andinhibit signalling induced by IL-4 and IL-13. Notably, the bindingmembers inhibit signalling from the high affinity complexes e.g.IL-4:IL-4Rα:γc, IL-4:IL-4Rα:IL-13Rα1, IL-13:IL-13Rα1:IL-4Rα. Such actionprevents signalling of both IL-4 and IL-13. Additionally, the dataindicate that the binding members inhibit interaction and signalling ofIL-4Rα type 1 and type 2 complexes. These and other properties andeffects of the binding members are described in further detail below.

The binding members are useful for treating disorders in which IL-4Rα,IL-4 or IL-13 are expressed, e.g., one or more of the IL-4Rα-, IL-4- orIL-13-related disorders referred to elsewhere herein, such as asthma orCOPD.

As described elsewhere herein, binding of a binding member to IL-4Rα maybe determined using surface plasmon resonance e.g. BIAcore.

Surface plasmon resonance data may be fitted to a 1:1 Langmuir bindingmodel (simultaneous ka kd) and an affinity constant KD calculated fromthe ratio of rate constants kd1/ka1. A binding member of the inventionmay have a monovalent affinity for binding human IL-4Rα that is lessthan 20 nM. In other embodiments the monovalent affinity for bindinghuman IL-4Rα that is less than 10 nM, e.g. less than 8, less than 5 nM.In other embodiments the binding member also binds cynomolgus IL-4Rα. Inone embodiment, a binding member of the present invention has amonovalent affinity for binding human IL-4Rα u) in the range 0.05 to 12nM. In one embodiment, a binding member of the present invention has amonovalent affinity for binding human IL-4Rα in the range of 0.1 to 5nM. In one embodiment, a binding member of the present invention has amonovalent affinity for binding human IL-4Rα in the range of 0.1 to 2nM.

In one embodiment, a binding member of the invention mayimmunospecifically bind to human IL-4Rα and may have an affinity (KD) ofless than 5000 pM, less than 4000 pM, less than 3000 pM, less than 2500pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than150 pM, less than 100 pM, less than 75 pM as assessed using a methoddescribed herein or known to one of skill in the art (e.g., a BIAcoreassay, ELISA) (Biacore International AB, Uppsala, Sweden).

In a specific embodiment, a binding member of the invention mayimmunospecifically bind to human IL-4Rα and may have an affinity (KD) ofbetween 25 to 5000 pM, 25 to 4000 pM, 25 to 3500 pM, 25 to 3000 pM, 25to 2500 pM, 25 to 2000 pM, 25 to 1500 pM, 25 to 1000 pM, 25 to 750 pM,25 to 500 pM, 25 to 250 pM, 25 to 100 pM, 25 to 75 pM, 25 to 50 pM asassessed using a method described herein or known to one of skill in theart (e.g., a BIAcore assay, ELISA). In another embodiment, ananti-IL-4Rα binding member (including an antibody) of the invention mayimmunospecifically bind to bind to human IL-4Rα and may have an affinity(KD) of 500 pM, 100 pM, 75 pM or 50 pM as assessed using a methoddescribed herein or known to one of skill in the art (e.g., a BIAcoreassay, ELISA).

As described in more detail in the Examples, binding members accordingto the invention neutralise IL-4Rα with high potency. Neutralisationmeans inhibition of a biological activity mediated by IL-4Rα. Bindingmembers of the invention may neutralise one or more activities mediatedby IL-4Rα. The inhibited biological activity is likely mediated byprevention of IL-4Rα forming a signalling complex with gamma chain (orIL-13Rα) and either of the associated soluble ligands, e.g. IL-4 orIL-13.

Neutralisation of IL-4 or IL-13 signalling through its IL-4Rα containingreceptor complex may be measured by inhibition of IL-4 or IL-13stimulated TF-1 cell proliferation.

The epitope of human IL4Rα to which the antibodies of the invention bindwas located by a combination of mutagenesis and domain swapping. Wholedomain swap chimeras localised the epitope to domain 1 (D1) of humanIL4Rα (residues M1-E119). Human IL-4Rα contains five loop regions, whichare in close proximity to IL4 in a crystal structure (Hage et al., Cell97:271-281, 1999). Loop swap chimeras enabled the further localisationof the human IL-4Rα epitope bound by an antibody of the invention, to amajor component in loop 3 (residues L89-N98) and a minor component inloop 2 (residues V65-H72). Chimeras without human loop 3 failed toinhibit human IL-4Rα binding to antibody and chimeras without loop 2gave a 100 fold higher IC₅₀ than human IL-4Rα (Table 5). Consistent withthe domain swap data both loop2 and loop3 are located in domain 1 (D1)(Hage et al., Cell 97:271-281, 1999).

The antibody epitope was located to a discontinuous epitope of 18 aminoacids in two loop regions of human IL-4Rα; V65-H72 and L89-N98. Theepitope can be further localised to amino acid residues L67 and L68 ofloop 2 and D92 and V93 of loop3 (see SEQ ID NO: 454 or 460 for locationof residues 67, 68, 92 and 93). The D92 residues was the most important,followed by V93, for the antibody tested was still capable of bindingchimeric IL-4Rα that lacked the L67 and/or L68 residues in loop2. Ofcourse it is likely that the antibodies of the invention will also bindresidues of the human IL-4Rα protein in addition to one of L67, L68, D92and V93.

According to one aspect of the invention there is provided an isolatedbinding member capable of binding to human interleukin-4 receptor alpha(hIL-4Rα) at least one amino acid residue selected from the amino acidat position 67, 68, 92 and 93, according to the position in SEQ ID NO:460. According to one aspect of the invention there is provided anisolated binding member capable of binding to at least one of amino acidresidues 67, 68, 92 and 93, according to the position in SEQ ID NO: 460,of native human interleukin-4 receptor alpha (hIL-4Rα). In a particularembodiment the isolated binding member is capable of binding to theamino acid at position 92 of hIL-4Rα, according to the position in SEQID NO: 460. In another embodiment the isolated binding member is capableof binding to D92 and at least one other residue selected from L67, L68and V93. In another embodiment the isolated binding member is capable ofbinding to D92 and V93. In another embodiment the isolated bindingmember is capable of binding to D92, V93 and either of L67 or L68. Inanother embodiment the antibody is capable of binding to each of L67,L68, D92 and V93. Each of these embodiments refers to amino acidpositions in hIL-4Rα whose locations can be identified according to thehIL-4Rα amino acid sequence (from positions 1-229) depicted in SEQ IDNO: 460. In one embodiment the binding member is able to bind to therecited epitope residues (i.e. at least one of positions 67, 68, 92 and93) of full-length hIL-4Rα. In one embodiment the binding member is ableto bind to the recited epitope residues (i.e. at least one of positions67, 68, 92 and 93) of native hIL-4Rα expressed on the cell surface. Inone embodiment the binding member is able to bind to the recited epitoperesidues (i.e. at least one of positions 67, 68, 92 and 93) ofrecombinantly expressed full-length (229 amino acid) hIL-4Rα.

According to a further aspect of the invention there is provided anisolated binding member capable of binding human interleukin-4 receptoralpha (hIL-4Rα). In a particular embodiment the binding member is ahuman antibody. In a further embodiment the binding member is alsocapable of binding cynomolgus monkey interleukin-4 receptor alpha(cyIL-4Rα).

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), which binding member has an IC₅₀ geomean for inhibition ofhuman IL-4 (hIL-4) induced cell proliferation of less than 50 pM in TF-1proliferation assay using 18 pM soluble human IL-4 protein and whichbinding member is also capable of binding cyIL-4Rα.

In particular embodiments of this aspect of the invention the bindingmember has an IC₅₀ geomean for inhibition of human IL-4 (hIL-4) inducedcell proliferation of less than 50 pM, less than 35 pM, less than 25 pM,or less than 20 pM, in a TF-1 proliferation assay using 18 pM of solublehuman IL-4. In particular embodiments the binding member of theinvention has an IC₅₀ geomean for inhibition of human IL-4 (hIL-4)induced cell proliferation of between 1 to 50 pM, 1 to 35 pM, 2 to 30pM, 2 to 25 pM, 2 to 12 pM, using 18 pM of soluble human IL-4 in amethod described herein (e.g. Example 3.2.1) or known to one of skill inthe art.

Binding to cyIL-4Rα can be measured by any suitable means.

Similarly, binding members within the scope of the invention have anIC₅₀ geomean for inhibition of human IL-13 (hIL-13)-mediated TF-1proliferation (via neutralisation of hIL-4Rα) of less than 200 pM using400 pM soluble human IL-13 (hIL-13). In a particular embodiment the IC₅₀geomean for inhibition of human IL-13 (hIL-13)-mediated TF-1proliferation (via neutralisation of hIL-4Rα) using 400 pM soluble humanIL-13 (hIL-13) is between 5 and 75 pM or between 5 and 45 pM.

In particular embodiments, the binding members of the invention aresubstantially incapable of binding to murine IL-4Rα. By this we meanthat a binding member of the invention is capable of at least 500-fold(such as at least 500-fold, at least 1000-fold, at least 1500-fold, atleast 2000-fold, at least 3000-fold, at least 4000-fold) greater bindingto human interleukin-4 receptor alpha than to murine IL-4Rα (i.e.binding to murine IL-4Rα is at least 500 fold weaker than to humanIL-4Rα). This can be measured, for example, by the HTRF competitionassay as disclosed in Example 5.1.2.

Geomean (also known as geometric mean), as used herein means the averageof the logarithmic values of a data set, converted back to a base 10number. This requires there to be at least two measurements, e.g. atleast 2, preferably at least 5, more preferably at least 10 replicate.The person skilled in the art will appreciate that the greater thenumber of replicates the more robust the geomean value will be. Thechoice of replicate number can be left to the discretion of the personskilled in the art.

Inhibition of biological activity may be partial or total. In specificembodiments, binding members are provided that inhibit IL-4Rα biologicalactivity by at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, or at least 50% of the activityin the absence of the binding member. The degree to which a bindingmember neutralises IL-4Rα is referred to as its neutralising potency.Potency may be determined or measured using one or more assays known tothe skilled person and/or as described or referred to herein. Forexample, potency may be assayed in:

Receptor-ligand binding assays in fluorescent (e.g. HTRF or DELFIA) orradioactive format

Fluorescent (e.g. HTRF or DELFIA) epitope competition assay

Cell-based functional assays including STAT6 Phosphorylation of human orcynomolgous PBMCs, proliferation of TF-1 cells, eotaxin release fromhuman or cynomolgous fibroblast cell lines, VCAM-1 upregulation on humanendothelial vein cells or proliferation of human T-cells.

Some of these assays methods are also described in the Examples.

Neutralising potency of a binding member as calculated in an assay usingIL-4Rα from a first species (e.g. human) may be compared withneutralising potency of the binding member in the same assay usingIL-4Rα from a second species (e.g. cynomolgus monkey), in order toassess the extent of cross-reactivity of the binding member for IL-4Rαof the two species. There are great advantages in having a bindingmember, e.g. an antibody or antibody fragment, which binds both thehuman target and the orthologous target from another species. A keyadvantage arises when the binding-member is being advanced as atherapeutic product and safety studies (e.g. toxicity) need to beconducted in another species. Potency or affinity to another species,which is for example less than 10-fold different than the human activitymay be appropriate for such an evaluation.

There are various ways of determining the ratio of binding of thebinding members of the present invention to the human and the “otherspecies” (e.g. cynomologus monkey) IL-4Rα. One method is thereceptor-ligand binding assay, such as that used in Example 4.3.

According to a particular embodiment of the binding members of thepresent invention the ratio of binding of the binding member when as ascFv to hIL-4Rα and to cyIL-4Rα measured using the receptor-ligandbinding assay is at least 6:1. As used here, “at least” 6:1, includes8:1, 10:1 etc; rather than 2:1, 1:1.

Binding members of the invention bind human IL-4Rα and cynomolgus monkeyIL-4Rα, and may have a less than 250-fold, e.g. less than 150-, 100-,75-, 50-, 25-, 20-, 15-, 10-fold difference in potency for neutralisinghuman and cynomolgus IL-4Rα as determined in the receptor-ligand bindingassay, with the binding member being in scFv format, as in Example 4.3.

For example, the data herein indicate that Antibody nos: 2, 4-8, 12, 16,19, 20, 22, 23, 24, 26, 28, 32, 33, 34, 37 and 37GL, for example, have aless than or equal to 25-fold difference in potency for neutralisinghuman and cynomolgus IL-4Rα respectively, when in scFv format in thereceptor-ligand binding assay described herein. Data are presented inExample 4.3 and Table 1. Thus, in some embodiments, neutralisationpotency of binding members of the invention (when in scFv format) forhuman and cynomolgus IL-4Rα measured using the receptor-ligand bindingassay is within 25-fold. Particular examples of antibodies describedherein that exhibit less than or equal to 10-fold neutralisation potencyfor human and cynomolgus IL-4Rα include Antibody nos 2, 4, 5, 20 and 22.In one embodiment the neutralisation potency of binding members of theinvention for human and cynomolgus IL-4Rα is within 210-fold; i.ebinding to human IL-4Rα is no greater than 210-fold that againstcynomologous IL-4Rα. In another embodiment, said neutralisation potencyis between 5:1 and 210:1, such as between 5:1 and 100:1.

For functional cell-based assays potency is normally expressed as anIC₅₀ value, in nM unless otherwise stated. In functional assays, IC₅₀ isthe molar concentration of a binding member that reduces a biological(or biochemical) response by 50% of its maximum. IC₅₀ may be calculatedby plotting % of maximal biological response as a function of the log ofthe binding member concentration, and using a software program such asPrism (GraphPad) or Origin (Origin Labs) to fit a sigmoidal function tothe data to generate IC₅₀ values. For receptor-ligand binding assays,potency is normally expressed as Ki (the inhibition constant), theconcentration of binding member that would occupy 50% of receptors if nolabelled ligand were present. Whereas IC₅₀ may vary between experimentsdepending on ligand concentration, the Ki is an absolute valuecalculated from the Cheng Prusoff equation.

A binding member of the invention may have a neutralising potency or Kiof up to 5 nM in a human IL-4RαHTRF® assay as described herein. Thisassay can be used to determine Ki for binding members in scFv format.The Ki may for example be up to 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.2, 0.1,0.05, or 0.02 nM. Examples of Ki data are presented in Example 4.3 (seeTable 1), wherein a final concentration of 0.125 nM human IL-4Rα and 2nM IL-4 is used in the HTRF® receptor-ligand binding assay and adetailed method is provided.

Additionally, binding kinetics and affinity (expressed as theequilibrium dissociation constant, KD) of IL-4Rα binding members forIL-4Rα may be determined, e.g. using surface plasmon resonance such asBIAcore®, or Kd may be estimated from pA₂ analysis.

Surface plasmon resonance is a well-established technique fordetermining affinity of a binding member for a target. It involvespassing an analyte in fluid phase over a ligand attached to a support,and determining binding between analyte and ligand. Surface plasmonresonance may for example be performed whereby recombinant IL-4Rα ispassed in fluid phase over a binding member attached to a support.Surface plasmon resonance data may be fitted to a bivalent analyte datamodel or a monovalent analyte data model. As shown in the Examplesherein, a monovalent analyte data model was found to be particularlyappropriate for determining affinity of binding members to IL-4Rα. Anaffinity constant Kd may be calculated from the ratio of rate constantskd1/ka1 as determined by surface plasmon resonance using a 1:1 Langmuirbinding model.

Examples of estimated KD values for binding IL-4Rα calculated usingsurface plasmon resonance are presented in Example 4.7 (see Table 4).These data demonstrate good binding properties of Antibody 37GL forrecombinantly produced human and cynomolgus IL-4Rα. Binding to IL-4Rαfrom HEK-EBNA cells demonstrates that the antibody binds nativeglycosylated human IL-4Rα. Because Antibody 37GL binds to the nativeglycosylated human IL-4Rα form allows one to predict that all ofantibodies described herein (e.g. Antibodies 1 to 42) are able to bindnative glycosylated human IL-4Rα, given that all these antibodies werederived from a single parent antibody (Antibody 1) and are thus believedto all bind the same or highly similar epitope of IL-4Rα.

Thus, according to particular embodiments, the binding members of theinvention are capable of binding to glycosylated hIL-4Rα.

As illustrated in Example 4.7 and Table 4, a good cross-reactivity inbinding human and cynomolgus IL-4Rα was determined by surface plasmonresonance for a representative panel of antibodies derived from Antibody1.

Binding members of the invention may optionally be specific for IL-4Rαover other structurally related molecules (e.g. other interleukinreceptors) and thus bind IL-4Rα selectively. For example, bindingmembers of the invention may not cross-react with any of IL-13Rα1 orIL-13Rα2 and the common gamma chain (γc). This may be determined ordemonstrated, for example, in a DELFIA® epitope competition assay asexemplified in Example 4.6.

A binding member of the invention may comprise an antibody molecule,e.g. a human antibody molecule. The binding member comprises an antibodyVH and/or VL domain. VH domains of binding members are also provided aspart of the invention. Within each of the VH and VL domains arecomplementarity determining regions, (“CDRs”), and framework regions,(“FRs”). A VH domain comprises a set of HCDRs, and a VL domain comprisesa set of LCDRs. An antibody molecule may comprise an antibody VH domaincomprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework. A VH or VL domain frameworkcomprises four framework regions, FR1, FR2, FR3 and FR4, interspersedwith CDRs in the following structure:

-   -   FR1—CDR1— FR2—CDR2— FR3—CDR3— FR4.

Examples of antibody VH and VL domains, FRs and CDRs according to thepresent invention are as listed in the appended sequence listing thatforms part of the present disclosure. All VH and VL sequences, CDRsequences, sets of CDRs and sets of HCDRs and sets of LCDRs u) disclosedherein represent aspects and embodiments of the invention. As describedherein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set ofHCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers toLCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “full set of CDRs”includes HCDRs and LCDRs. Typically, binding members of the inventionare monoclonal antibodies.

A further aspect of the invention is an antibody molecule comprising aVH domain that has at least 75, 80, 85, 90, 95, 98 or 99% amino acidsequence identity with a VH domain of any of Antibodies 1 to 42 shown inthe appended sequence listing, and/or comprising a VL domain that has atleast 75, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with aVL domain of any of Antibodies 1 to 42 shown in the appended sequencelisting. Accelerys' “MacVector™” program may be used to calculate %identity of two amino acid sequences.

A binding member of the invention may comprise an antigen-binding sitewithin a non-antibody molecule, normally provided by one or more CDRse.g. an HCDR3 and/or LCDR3, or a set of CDRs, in a non-antibody proteinscaffold, as discussed further below.

As described in more detail in the Experimental Section, the inventorsisolated a parent antibody molecule (Antibody 1) with a set of CDRsequences as shown in FIGS. 1 (VH domain) and 2 (VL domain). Through aprocess of optimisation they generated a panel of antibody clones,including those numbered 2 to 20, with CDR3 sequences derived from theparent CDR3 sequences and having substitutions at the positionsindicated in FIG. 1 (VH domain) and FIG. 2 (VL domain). Thus forexample, it can be seen from FIG. 1 (a and b), that Antibody 2 has theparent HCDR1, HCDR2, LCDR1 and LCDR2 sequences, and has the parent LCDR3sequence in which Kabat residue 95 is replaced by Q, Kabat residue 95A,95B and 96 are each replaced by P and Kabat residue 97 is replaced by L;and has parent HCDR3 sequence in which Kabat residue 101 is replaced byY and Kabat residue 102 is replaced by N.

The parent antibody molecule, and Antibody molecules 2 to 20, asdescribed herein refer respectively to antibody molecules with CDRs ofthe parent antibody molecule and to antibody molecules with CDRs ofantibody molecules 2 to 20. Through a further process of optimisationthe inventors generated a panel of antibody clones numbered 21-42, withadditional substitutions throughout the VH and VL domains. Thus, forexample, Antibody 21 has the same LCDR1, LCDR2, LCDR3, HCDR1, and HCDR3as Antibody 20; it has the parent HCDR2 sequence of Antibody 20 but withKabat residue 57 replaced by A; and it has Kabat residues 85 and 87 (inLFW3) replaced by V and F, respectively.

Described herein is a reference binding member comprising the Antibody20 set of CDRs as shown in FIGS. 3 (VH) and 4 (VL), in which HCDR1 isSEQ ID NO: 193 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 194 (Kabatresidues 50-65), HCDR3 is SEQ ID NO: 195 (Kabat residues 95-102), LCDR1is SEQ ID NO: 198 (Kabat residues 24-34), LCDR2 is SEQ ID NO: 199 (Kabatresidues 50-56) and LCDR3 is SEQ ID NO: 200 (Kabat residues 89-97).Further binding members can be described with reference to the sequencein the reference binding member.

A binding member of the invention may comprise one or more CDRs (i.e. atleast one, at least 2, at least 3, at least 4 at least 5 and at least 6)as described herein, e.g. a CDR3, and optionally also a CDR1 and CDR2 toform a set of CDRs. The CDR or set of CDRs may be a parent CDR or parentset of CDRs, or may be a CDR or set of CDRs of any of Antibodies 2 to42, or may be a variant thereof as described herein.

For example, a binding member or a VL domain according to the inventionmay comprise the reference LCDR3 with one or more of Kabat residues92-97 substituted for another amino acid. Exemplary substitutionsinclude:

Kabat residue 92 replaced by Phe (F), Val (V) or Ala (A);Kabat residue 93 replaced by Gly (G) or Ser (S);Kabat residue 94 replaced by Thr (T)Kabat residue 95 replaced by Leu (L), GLn (Q), Pro (P) or Ser (S);Kabat residue 95a replaced by Ser (S), Pro1 (P), Ala (A), Thr (T), His(H) or Gly (G);Kabat residue 95b replaced by Ala (A), Pro (P), Ser (S), Tyr (Y), Met(M), Leu (L), Thr (T), Arg (R) or Asp (D);Kabat residue 95c replaced by Asn (N), Gln (Q), H is (H), Tyr (Y), Thr(T), Ile (I), Lys (K), Arg (R) or Met (M);Kabat residue 96 replaced by Tyr (Y) or Pro (P);Kabat residue 97 replaced by Val (V), Leu (L) or Ile (I).

A binding member or a VH domain may comprise the reference HCDR3 withone or more of Kabat residues 97-102 substituted for another amino acid.Exemplary substitutions include:

Kabat residue 97 replaced by Trp (W) or Leu (L);Kabat residue 98 replaced by Leu (L);Kabat residue 99 replaced by Leu (L), Lys (K), Phe (F) or Trp (W);Kabat residue 101 replaced by Asp (D), Asn (N) or Gln (Q);Kabat residue 102 replaced by Tyr (Y), Asn (N), Pro (P) or His (H).

Binding members of the invention may comprise an HCDR1, HCDR2 and/orHCDR3 of any of Antibodies 1 to 42 and/or an LCDR1, LCDR2 and/or LCDR3of any of Antibodies 1 to 42, e.g. a set of CDRs of any of Antibodies 1to 42 shown in FIG. 1 or 2. A binding member may comprise a set of VHCDRs of one of these antibodies. Optionally it may also comprise a setof VL CDRs of one of these antibodies, and the VL CDRs may be from thesame or a different antibody as the VH CDRs. A VH domain comprising aset of HCDRs of any of Antibodies 1 to 42, and/or a VL domain comprisinga set of LCDRs of any of Antibodies 1 to 42, are also individualembodiments of the invention.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH or VLdomain alone may be used to bind antigen. In one embodiment, theAntibody 1 VH domain is paired with the Antibody 1 VL domain, so that anantibody antigen-binding site is formed comprising both the Antibody 1VH and VL domains. Analogous embodiments are provided for the other VHand VL domains disclosed herein. In other embodiments, the Antibody 1 VHis paired with a VL domain other than the antibody 1 VL. Light-chainpromiscuity is well established in the art. Again, analogous embodimentsare provided by the invention for the other VH and VL domains disclosedherein. Thus, the VH of the parent (Antibody 1) or of any of Antibodies2 to 42 may be paired with the VL of the parent or of any of Antibodies2 to 42.

One aspect of the invention is an antibody comprising a VH and VL domainwherein the VH domain comprises a sequence disclosed in FIG. 1 or 3.

Another aspect of the invention is an antibody comprising a VH and VLdomain wherein the VL domain comprises a sequence disclosed in FIG. 2 or4.

Another aspect of the invention is an isolated antibody moleculecomprising a VH domain with the VH domain amino acid sequence shown inSEQ ID NO: 362, 442, 232, 422 or 432 and a VL domain with the VL domainamino acid sequence shown in SEQ ID NOs: 367, 237, 447, 437 or 427

A binding member may comprise a set of H and/or L CDRs of the parentantibody or any of Antibodies 2 to 42 with twelve or ten or nine orfewer, e.g. one, two, three, four or five, substitutions within thedisclosed set of H and/or L CDRs. For example, a binding member of theinvention may comprise the Antibody 16 or Antibody 20 set of H and/or LCDRs with 12 or fewer substitutions, e.g. seven or fewer substitutions,e.g. zero, one, two, three, four, five, or six substitutions.Substitutions may potentially be made at any residue within the set ofCDRs, and may be within CDR1, CDR2 and/or CDR3.

Thus, according to one aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs:

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the set of CDRs has12 or fewer amino acid alterations from a reference set of CDRs inwhich:HCDR1 has amino acid sequence SEQ ID NO: 153;HCDR2 has amino acid sequence SEQ ID NO: 154;HCDR3 has amino acid sequence SEQ ID NO: 155;LCDR1 has amino acid sequence SEQ ID NO: 158;LCDR2 has amino acid sequence SEQ ID NO: 159; andLCDR3 has amino acid sequence SEQ ID NO: 160.The reference antibody in this instance is Antibody 16.

The isolated binding member may have 10 or fewer, 8 or fewer, 7 orfewer, e.g. 6, 5, 4, 3, 2, 1 or 0 amino acid alterations from thereference set of CDRs. Particular alterations are amino acidsubstitutions.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, wherein the set of CDRs has 12 or fewer amino acidalterations from a reference set of CDRs in which:

HCDR1 has amino acid sequence SEQ ID NO: 193;HCDR2 has amino acid sequence SEQ ID NO: 194;HCDR3 has amino acid sequence SEQ ID NO: 195;LCDR1 has amino acid sequence SEQ ID NO: 198;LCDR2 has amino acid sequence SEQ ID NO: 199; andLCDR3 has amino acid sequence SEQ ID NO: 200.The reference antibody in this instance is Antibody 20.

The isolated binding member may have 10 or fewer, 8 or fewer, 7 or fewere.g. 6, 5, 4, 3, 2, 1 or 0 amino acid alterations from the reference setof CDRs. Particular alterations are amino acid substitutions. In aparticular embodiment, the isolated binding member has 4 or fewer aminoacid substitutions from the reference set of CDRs identified above.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, wherein the set of CDRs has 6 or fewer amino acid alterationsfrom a reference set of CDRs in which:

HCDR1 has amino acid sequence SEQ ID NO: 363;HCDR2 has amino acid sequence SEQ ID NO: 364;HCDR3 has amino acid sequence SEQ ID NO: 365;LCDR1 has amino acid sequence SEQ ID NO: 368;LCDR2 has amino acid sequence SEQ ID NO: 369; andLCDR3 has amino acid sequence SEQ ID NO: 370.The reference antibody in this instance is Antibody 37.

Substitutions may be within CDR3, e.g. at the positions substituted inany of Antibodies 2 to 42, as shown in FIG. 1 or 3 (VH domain) and 2 or4 (VL domain). Thus, the one or more substitutions may comprise one ormore substitutions at the following residues:

Kabat residue 97, 98, 99, 101 or 102 in HCDR3; orKabat residue 92, 93, 94, 95, 95A, 95B, 95C, 96 or 97 in LCDR3.

Thus, a CDR3 may for example be a reference LCDR3 having one or moresubstitutions at Kabat residues 92, 93, 94, 95, 95A, 95B, 95C, 96 or 97.

Examples of substitutions in parent/reference CDRs are describedelsewhere herein. As described, the substitutions may comprise one ormore substitutions as shown in FIGS. 1 to 4.

A binding member of the invention may comprise the HCDR1, HCDR2 and/orHCDR3 of the reference Antibody 20, or with one or more of the followingsubstitutions:

HCDR2 wherein Kabat residue 53 is Arg (R);HCDR2 wherein Kabat residue 57 is Ala (A);HCDR3 wherein Kabat residue 97 is Trp (W) or Leu (L); Kabat residue 98is Leu; Kabat residue 99 is Leu (L), Lys (K) or Trp (W); Kabat residue101 is Asn (N) or Gln (Q); and/or Kabat residue 102 is Tyr (Y), Asn (N),Pro (P) or His (H).

A binding member of the invention may comprise an LCDR1, LCDR2 and/orLCDR3 of the reference Antibody 20, or with one or more of the followingsubstitutions:

LCDR1 wherein Kabat residue 27 is Gly (G);

Kabat residue 27A is Thr (T);Kabat residue 27B is Ser (S);Kabat residue 31 is Asn (N);LCDR2 wherein Kabat residue 56 is Pro (P);

LCDR3 wherein Kabat residue 92 is Phe (F), Val (V) or Ala (A);

Kabat residue 93 is Gly (G) or Ser (S);Kabat residue 94 is Thr (T);Kabat residue 95 is Leu (L), Gln (O), Pro (P) or Ser (S);Kabat residue 95A is Ser (S), Pro (P), Ala (A), Thr (T), His (H) or Gly(G);

Kabat residue 95B is Ala (A), Pro (P), Ser (S), Tyr (Y), Met (M), Leu(L), Thr (T), Asp (D) or Arg (R);

Kabat residue 95C is Asn (N), Gln (Q), His (H), Tyr (Y), Ile (I), Lys(K), Arg (R), Thr (T) or Met (M);Kabat residue 96 is Tyr (Y) or Pro (P);and/or Kabat residue 97 is Val (V), Leu (L) or Ile (I).

In a particular embodiment, with reference to Antibody 20 sequence,Kabat residue 53 in HCDR2 is replaced by Arg (R);

and/or Kabat residue 57 in HCDR2 is replaced by Ala (A);and/or Kabat residue 27 in LCDR1 is replaced by Gly (G); and/or Kabatresidue 27B in LCDR1 is replaced by Ser (S); and/or Kabat residue 95 inLCDR3 is replaced by Pro (P).

According to a particular aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), wherein the HCDR1 has amino acid sequence SEQ ID NO: 363;

the HCDR2 has amino acid sequence SEQ ID NO: 364;the HCDR3 has amino acid sequence SEQ ID NO: 365;the LCDR1 has amino acid sequence SEQ ID NO: 368;the LCDR2 has amino acid sequence SEQ ID NO: 369; andthe LCDR3 has amino acid sequence SEQ ID NO: 370;

According to another particular aspect of the invention there isprovided an isolated binding member for human interleukin-4 receptoralpha (hIL-4Rα), wherein the HCDR1 has amino acid sequence SEQ ID NO:233;

the HCDR2 has amino acid sequence SEQ ID NO: 234;the HCDR3 has amino acid sequence SEQ ID NO: 235;the LCDR1 has amino acid sequence SEQ ID NO: 238;the LCDR2 has amino acid sequence SEQ ID NO: 239; andthe LCDR3 has amino acid sequence SEQ ID NO: 240;

In a binding member of the invention:

HCDR1 may be 5 amino acids long, consisting of Kabat residues 31-35;HCDR2 may be 17 amino acids long, consisting of Kabat residues 50-65;HCDR3 may be 9 amino acids long, consisting of Kabat residues 95-102;LCDR1 may be 13 amino acids long, consisting of Kabat residues 24-34;LCDR2 may be 7 amino acids long, consisting of Kabat residues 50-56;and/or, LCDR3 may be 9 amino acids long, consisting of Kabat residues89-97.

Kabat numbering of a set of HCDRs and LCDRs, wherein HCDR1 is Kabatresidues 31-35, HCDR2 is Kabat residues 50-65, HCDR3 is Kabat residues95-102 is shown in FIGS. 1 and 3; LCDR1 is Kabat residues 24-34, LCDR2is Kabat residues 50-56 and LCDR3 is Kabat residues 89-97, is shown inFIGS. 2 and 4.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, wherein the set of CDRs has 6 or fewer amino acid alterationsfrom the reference set of CDRs present in the clone deposited at NCIMBon 9^(th) Dec. 2008 with accession number: NCIMB 41600.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a VH sequence as found in the clone deposited atNCIMB on 9 Dec. 2008 with accession number: NCIMB 41600.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a VL sequence as found in the clone deposited atNCIMB on 9^(th) December 2008 with accession number: NCIMB 41600.

According to another aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a VH and VL sequence as found in the clonedeposited at NCIMB on 9 Dec. 2008 with accession number: NCIMB 41600.

According to another aspect of the invention there is provided anisolated antibody or fragment of an antibody, wherein the antibody orthe fragment immunospecifically binds to human interleukin-4 receptoralpha and comprises:

(a) a VH CDR1 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VH CDR1present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600;

(b) a VH CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VH CDR2present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600;

(c) a VH CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VH CDR3present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600;(d) a VL CDR1 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VL CDR1present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600;(e) a VL CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VL CDR2present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600; and(f) a VL CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to the VL CDR3present in the clone deposited at NCIMB on 9 Dec. 2008 with accessionnumber: NCIMB 41600.

According to another aspect of the invention there is provided anisolated antibody or fragment of an antibody, wherein the antibody orthe fragment immunospecifically binds to human interleukin-4 receptoralpha and comprises:

(a) a VH sequence having an amino acid sequence identical to orcomprising 1, 2, 3, 4, 5, or 6 amino acid residue substitutions relativeto the VH sequence present in the clone deposited at NCIMB on 9 Dec.2008 with accession number: NCIMB 41600;(b) a VL sequence having an amino acid sequence identical to orcomprising 1, 2, 3, 4, 5, or 6 is amino acid residue substitutionsrelative to the VL sequence present in the clone deposited at NCIMB on9^(th) Dec. 2008 with accession number: NCIMB 41600.

A binding member may comprise an antibody molecule having one or moreCDRs, e.g. a set of CDRs, within an antibody framework. For example, oneor more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule.Framework regions may comprise human germline gene segment sequences.Thus, the framework may be germlined, whereby one or more residueswithin the framework are changed to match the residues at the equivalentposition in the most similar human germline framework. The skilledperson can select a germline segment that is closest in sequence to theframework sequence of the antibody before germlining and test theaffinity or activity of the antibodies to confirm that germlining doesnot significantly reduce antigen binding or potency in assays describedherein. Human germline gene segment sequences are known to those skilledin the art and can be accessed for example from the VBase compilation(see Tomlinson. Journal of Molecular Biology. 224. 487-499, 1997).

In one embodiment, a binding member of the invention is an isolatedhuman antibody molecule having a VH domain comprising a set of HCDRs ina human germline framework, e.g. Vh1_DP-7_(1-46). Thus, the VH domainframework regions FR1, FR2 and/or FR3 may comprise framework regions ofhuman germline gene segment Vh1_DP-7_(1-46). FR4 may comprise aframework region of human germline j segment JH1, JH4 or JH5 (these jsegments have identical amino acid sequences) or it may comprise aframework region of human germline j segment JH3. The amino acidsequence of VH FR1 may be SEQ ID NO: 442 (residues 1-30). The amino acidsequence of VH FR2 may be SEQ ID NO: 442 (residues 36-49). The aminoacid sequence of VH FR3 may be SEQ ID NO: 442 (residues 66-94). Theamino acid sequence of VH FR4 may be SEQ ID NO: 442 (103-113). Normallythe binding member also has a VL domain comprising a set of LCDRs, e.g.in a human germline framework, e.g. Vλ1_DPL5. Thus, the VL domainframework regions FR1, FR2 and/or FR3 may comprise framework regions ofhuman germline gene segment Vλ1 DPL5. FR4 may comprise a frameworkregion of human germline j segment JL2 or JL3 (these j segments haveidentical amino acid sequences). The amino acid sequence of VL FR1 maybe SEQ ID NO: 447 (residues 1-23). The amino acid sequence of VL FR2 maybe SEQ ID NO: 447 (residues 35-49). The amino acid sequence of VL FR3may be SEQ ID NO: 447 (residues 57-88). The amino acid sequence of VLFR4 may be SEQ ID NO: 447 (residues 98-107). A germlined VH or VL domainmay or may not be germlined at one or more Vernier residues, but isnormally not.

An antibody molecule or VH domain of the invention may comprise thefollowing set of heavy chain framework regions:

FR1SEQ ID NO: 442 (residues 1-30);FR2SEQ ID NO: 442 (residues 36-49);FR3SEQ ID NO: 442 (residues 66-94);FR4SEQ ID NO: 442 (residues 103-113); or may comprise the said set ofheavy chain framework regions with one, two, three, four, five, or sixamino acid alterations, e.g. substitutions.

An antibody molecule or VL domain of the invention may comprise thefollowing set of light chain framework regions:

FR1SEQ ID NO: 447 (residues 1-23);FR2SEQ ID NO: 447 (residues 35-49);FR3SEQ ID NO: 447 (residues 57-88);FR4SEQ ID NO: 447 (residues 98-107); or may comprise the said set ofheavy chain framework regions with one, two, three, four, five, or sixamino acid alterations, e.g. substitutions.

An amino acid alteration may be a substitution, an insertion (addition)or a deletion.

The most common alteration is likely to be a substitution.

For example, an antibody molecule of the invention may comprise a set ofheavy and light chain framework regions, wherein:

heavy chain FR1 is SEQ ID NO: 192(residues 1-30);heavy chain FR2 is SEQ ID NO: 192 (residues 36-49);heavy chain FR3 is SEQ ID NO: 192 (residues 66-94);heavy chain FR4 is SEQ ID NO: 192 (residues 103-113);light chain FR1 is SEQ ID NO: 197 (residues 1-23);light chain FR2 is SEQ ID NO: 197 (residues 35-49);light chain FR3 is SEQ ID NO: 197(residues 57-88);light chain FR4 is SEQ ID NO: 197 (residues 98-107);or may comprise thesaid set of heavy and light chain framework regions with seven or fewer,e.g. six or fewer, amino acid alterations, e.g. substitutions. Forexample there may be one or two amino acid substitutions in the said setof heavy and light chain framework regions.

As indicated in Example 4.2, Antibodies 21-42 are based on Antibody 20,but with certain additional alterations within the CDRs and frameworkregions. Like Antibody 20, Antibodies 21-42 bind hIL-4Rα and cyIL-4Rα.Such CDR and/or framework substitutions may therefore be considered asoptional or additional substitutions generating binding members withpotentially greater binding.

Thus, in addition to the substitutions within any of the 6 CDR regionsof the VH and VL domains, the binding members may also comprise one ormore amino acid substitutions at the following residues within theframework regions, using the standard numbering of Kabat:

11, 12 in HFW1; 37, 48 in HFW2; 68, 84, 85 in HFW3; 105, 108, 113 inHFW4;

1, 2, 3, 9 in LFW1;

38, 42 in LFW2; or 58, 65, 66, 70, 74, 85, 87 in LFW3.

Suitable framework substitutions are shown in FIGS. 1 to 4. And abinding member of the present invention may comprise one or more of thespecific substitutions shown in FIGS. 1 to 4.

An antibody molecule or VH domain of the invention may comprise a VH FR1wherein Kabat residue 11 is Val or Glu and/or Kabat residue 12 is Lys orArg; An antibody molecule or VH domain of the invention may comprise aVH FR2 wherein Kabat residue 37 is Ala or Val and/or Kabat residue 48 isMet or Val; An antibody molecule or VH domain of the invention maycomprise a VH FR3 wherein Kabat residue 68 is Ser, Ala or Thr and/orKabat residue 84 is Ser or Pro and/or Kabat residue 85 is Glu or Gly; Anantibody molecule or VH domain of the invention may comprise a VH FR4wherein Kabat residue 105 is Lys or Asn and/or Kabat residue 108 is Gln,Arg or Leu and/or Kabat residue 113 is Ser or Gly.

An antibody molecule or VL domain of the invention may comprise a VL FR1wherein Kabat residue 1 is Gln or Leu and/or Kabat residue 2 is Ser orPro or Ala and/or Kabat residue 3 is Val or Ala and/or Kabat residue 9is Ser or Leu; An antibody molecule or VL domain of the invention maycomprise a VL FR2 wherein Kabat residue 38 is Gln or Arg and/or Kabatresidue 42 is Thr or Ala; An antibody molecule or VL domain of theinvention may comprise a VL FR3 wherein Kabat residue 58 is Ile or Valand/or Kabat residue 65 is Ser or Phe and/or Kabat residue 66 is Lys orArg and/or Kabat residue 70 is Ser or Thr and/or Kabat residue 74 is Alaor Gly and/or Kabat residue 85 is Asp or Val and/or Kabat residue 87 isTyr or Phe.

A non-germlined antibody has the same CDRs, but different frameworks,compared with a germlined antibody. Of the antibody sequences shownherein, VH and VL domains of Antibodies 24PGL and 37GL are germlined.

FIGS. 5, 6 and 7, depict the composite sequence identity that each ofAntibodies 1-42 have with each other. The composite sequence being anartificial alignment of key CDR regions. Thus, for FIG. 5, the 6 CDRdomains (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3; 6xCDR) have beenaligned such that the last codon of LCDR1 is adjacent to the first codonof LCDR2, the last codon of LCDR2 is adjacent the first codon of LCDR3etc. In this manner a sequence that lacks the intervening frameworkregions is created. The composite sequence can be of all CDR regions, asfor FIG. 5, or just the heavy or light chain CDR sequences (as for FIGS.6 and 7 respectively). Sequence alignment of each Antibody compositesequence to each other Antibody composite sequence can then be generatedand the identity score depicted in the charts of FIGS. 5, 6 and 7. Ascan be seen from FIG. 5, with respect to all 6 CDR regions, the lowestdegree of sequence identity that any of Antibodies 1-42 has to anotheris 73% (this being Antibody 3 compared to Antibodies 23, 25, 37, 37GLand 41); if you exclude Antibody 3 the degree of sequence identity is78%. The lowest degree of sequence identity comparing only the threeHeavy chain CDR (3×HCDR) regions is 75% and 79% if you exclude Antibody3. The lowest degree of sequence identity comparing only the three Lightchain CDR (3xLCDR) regions is 65%; and, 75% if you exclude Antibody 3.

In a particular embodiment the isolated binding member has at least 73%amino acid sequence identity with the 6xCDR composite score of any ofAntibodies 1-42.

According to a further aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, which binding member has at least 73% amino acid sequenceidentity with the composite sequence of HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3 in line sequence without any intervening frameworksequences, of any of Antibodies 1-42. In a particular embodiment theisolated binding member has at least 78% amino acid sequence identitywith the composite score of any of Antibodies 1-42.

According to a further aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, which binding member has at least 75% amino acid sequenceidentity with the composite sequence of HCDR1, HCDR2 and HCDR3 of any ofAntibodies 1-42.

According to a further aspect of the invention there is provided anisolated binding member for human interleukin-4 receptor alpha(hIL-4Rα), comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, which binding member has at least 65% amino acid sequenceidentity with the composite sequence of LCDR1, LCDR2 and LCDR3 of any ofAntibodies 1-42.

A binding member of the present invention may be one which competes forbinding to IL-4Rα with any binding member which both binds IL-4Rα andcomprises a binding member, VH and/or VL domain, CDR e.g. HCDR3, and/orset of CDRs disclosed herein. Competition between binding members may beassayed easily in vitro, for example using ELISA and/or by tagging aspecific reporter molecule to one binding member which can be detectedin the presence of one or more other untagged binding members, to enableidentification of binding members which bind the same epitope or anoverlapping epitope. Competition may be determined for example usingELISA in which IL-4Rα is immobilized to a plate and a first taggedbinding member along with one or more other untagged binding members isadded to the plate. Presence of an untagged binding member that competeswith the tagged binding member is observed by a decrease in the signalemitted by the tagged binding member. Such methods are readily known toone of ordinary skill in the art, and are described in more detailherein. In one embodiment, competitive binding is assayed using anepitope competition assay as described herein. A binding member of thepresent invention may comprise a antibody antigen-binding site thatcompetes with an antibody molecule, for example especially an antibodymolecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRsof the parent antibody or any of Antibodies 2 to 42 for binding toIL-4Rα.

Aspects of the invention provide binding members that compete forbinding to IL-4Rα with any binding member defined herein, e.g. competewith the parent antibody or any of Antibodies 2 to 42, e.g. in scFv orIgG1, IgG2 or IgG4 format. A binding member that competes for binding toIL-4Rα with any binding member defined herein may have any one or moreof the structural and/or functional properties disclosed herein forbinding members of the invention.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a binding member, such as a VHdomain and/or VL domain according to the present invention, and methodsof preparing a binding member, such as a VH domain and/or a VL domain ofthe invention, which comprise expressing said nucleic acid underconditions to bring about production of said binding member, such as aVH domain and/or VL domain and/or antibody, and recovering it.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein.

A further aspect provides a host cell containing or transformed withnucleic acid of the invention.

Further aspects of the present invention provide for compositionscontaining binding members of the invention, and their use in methods ofinhibiting and/or neutralising IL-4Rα, including methods of treatment ofthe human or animal body by therapy.

Binding members according to the invention may be used in a method oftreatment or diagnosis of the human or animal body, such as a method oftreatment (which may include prophylactic treatment) of a disease ordisorder in a human patient which comprises administering to saidpatient an effective amount of a binding member of the invention.Conditions treatable in accordance with the present invention includeany in which IL-4Rα, IL-4 and/or IL-13 plays a role, as discussed indetail elsewhere herein. These and other aspects of the invention aredescribed in further detail below.

Terminology

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

IL-4Rα

IL-4Rα, is interleukin-4 receptor alpha. References to IL-4Rα arenormally to human IL-4Rα is unless otherwise indicated. A sequence ofwild-type mature human IL-4Rα is deposited under Accession number P24394(Swiss-Prot), which shows the full-length IL-4Rα including the signalpeptide.

Cynomolgus IL-4Rα was sequenced in house, the cDNA sequence ofcynomolgus IL-4Rα is shown as SEQ ID NO: 455.

As described elsewhere herein, IL-4Rα may be recombinant, and/or may beeither glycosylated or unglycosylated. IL-4Rα is expressed naturally invivo in N-linked glycosylated form. Glycosylated IL-4Rα may also beexpressed in recombinant systems, e.g. in HEK-EBNA cells. IL-4Rα mayalso be expressed in non-glycosylated form in E. coli cells.

Binding Member

This describes one member of a pair of molecules that bind one another.The members of a binding pair may be naturally derived or wholly orpartially synthetically produced. One member of the pair of moleculeshas an area on its surface, or a cavity, which binds to and is thereforecomplementary to a particular spatial and polar organization of theother member of the pair of molecules. Examples of types of bindingpairs are antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is concernedwith antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen-bindingsite. For example, a binding member may be an antibody molecule or anon-antibody protein that comprises an antigen-binding site.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds such as fibronectin or cytochrome Betc. (Haan & Maggos, BioCentury, 12(5):A1-A6, 2004; Koide, Journal ofMolecular Biology, 284:1141-1151, 1998; Nygren et al., Current Opinionin Structural Biology, 7:463-469, 1997), or by randomising or mutatingamino acid residues of a loop within a protein scaffold to conferbinding specificity for a desired target. Scaffolds for engineeringnovel binding sites in proteins have been reviewed in detail by Nygrenet al. (supra). Protein scaffolds for antibody mimics are disclosed inWO/0034784, which is herein incorporated by reference in its entirety,in which the inventors describe proteins (antibody mimics) that includea fibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDRs, e.g. a set ofHCDRs or an HCDR and/or LCDR3, may be provided by any domain member ofthe immunoglobulin gene superfamily. The scaffold may be a human ornon-human protein. An advantage of a non-antibody protein scaffold isthat it may provide an antigen-binding site in a scaffold molecule thatis smaller and/or easier to manufacture than at least some antibodymolecules. Small size of a binding member may confer usefulphysiological properties such as an ability to enter cells, penetratedeep into tissues or reach targets within other structures, or to bindwithin protein cavities of the target antigen. Use of antigen bindingsites in non-antibody protein scaffolds is reviewed in Wess, 2004.Typical are proteins having a stable backbone and one or more variableloops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g.10th fibronectin type III domain) and lipocalins. Other approachesinclude synthetic “Microbodies” (Selecore GmbH), which are based oncyclotides—small proteins having intra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, abinding member according to the present invention may comprise otheramino acids, e.g. forming a peptide or polypeptide, such as a foldeddomain, or to impart to the molecule another functional characteristicin addition to ability to bind antigen. Binding members of the inventionmay carry a detectable label, or may be conjugated to a toxin or atargeting moiety or enzyme (e.g. via a peptidyl bond or linker). Forexample, a binding member may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by non-antibody scaffolds, thestructure for carrying a CDR, e.g. CDR3, or a set of CDRs of theinvention will generally be an antibody heavy or light chain sequence orsubstantial portion thereof in which the CDR or set of CDRs is locatedat a location corresponding to the CDR or set of CDRs of naturallyoccurring VH and VL antibody variable domains encoded by rearrangedimmunoglobulin genes. The structures and locations of immunoglobulinvariable domains may be determined by reference to Kabat (Sequences ofProteins of Immunological Interest, 4^(th) Edition. US Department ofHealth and Human Devices, 1987), and updates thereof, such as the 5^(th)Edition (Sequences of Proteins of Immunological Interest, 5^(th)Edition. US Department of Health and Human Services, Public Service,NIH, Washington, 1991).

Unless indicated otherwise, the locations of particular residues, aswell as CDR and framework regions, referred to herein uses the Kabatnumbering system.

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al., (supra). An antibody typically contains 3 heavy chainCDRs and 3 light chain CDRs. The term CDR or CDRs is used here in orderto indicate, according to the case, one of these regions or several, oreven the whole, of these regions which contain the majority of the aminoacid residues responsible for the binding by affinity of the antibodyfor the antigen or the epitope which it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has greater size variability (greater diversity essentially dueto the mechanisms of arrangement of the genes which give rise to it). Itcan be as short as 2 amino acids although the longest size known is 26.Functionally, HCDR3 plays a role in part in the determination of thespecificity of the antibody (Segal et al. PNAS, 71:4298-4302, 1974; Amitet al., Science, 233:747-753, 1986; Chothia et al. J. Mol. Biol.,196:901-917, 1987; Chothia et al. Nature, 342:877-883, 1989; et al. J.Immunol., 144:1965-1968, 1990; Sharon et al. PNAS, 87:4814-4817,1990(a); Sharon et al. J. Immunol., 144:4863-4869, 1990; Kabat et al.,et al., J. Immunol., 147:1709-1719, 1991b).

HCDR1 may be 5 amino acids long, consisting of Kabat residues 31-35.HCDR2 may be 17 amino acids long, consisting of Kabat residues 50-65.HCDR3 may be 7 amino acids long, consisting of Kabat residues 95-102.LCDR1 may be 13 amino acids long, consisting of Kabat residues 24-34.LCDR2 may be 7 amino acids long, consisting of Kabat residues 50-56.LCDR3 may be 12 amino acids long, consisting of Kabat residues 89-97.

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody antigen-binding site. It must be understood herethat the invention does not relate to the antibodies in natural form,that is to say they are not in their natural environment but that theyhave been able to be isolated or obtained by purification from naturalsources, or else obtained by genetic recombination, or by chemicalsynthesis, and that they can then contain unnatural amino acids as willbe described later. Antibody fragments that comprise an antibodyantigen-binding site include, but are not limited to molecules such asFab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd; and diabodies.

Antibody molecules of the invention may be IgG, e.g. IgG1, IgG4, IgG2 oraglycosyl IgG2.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that bind the target antigen. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe CDRs, of an antibody to the constant regions, or constant regionsplus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site with the requiredspecificity and/or binding to antigen. Thus, this term covers antibodyfragments and derivatives, including any polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antibody antigen-bindingsite, or equivalent, fused to another polypeptide (e.g. derived fromanother species or belonging to another antibody class or subclass) aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel(Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN:3540413545). Phage display, another established technique for generatingbinding members has been described in detail in many publications suchas WO92/01047 (discussed further below) and U.S. Pat. No. 5,969,108,U.S. Pat. No. 5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat. No.5,858,657, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,872,215, U.S. Pat.No. 5,885,793, U.S. Pat. No. 5,962,255, U.S. Pat. No. 6,140,471, U.S.Pat. No. 6,172,197, U.S. Pat. No. 6,225,447, U.S. Pat. No. 6,291,650,U.S. Pat. No. 6,492,160, U.S. Pat. No. 6,521,404 and Kontermann & Dubel(supra). Transgenic mice in which the mouse antibody genes areinactivated and functionally replaced with human antibody genes whileleaving intact other components of the mouse immune system, can be usedfor isolating human antibodies (Mendez et al. Nature Genet,15(2):146-156, 1997).

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.(J. Mol. Biol. 296, 57-86, 2000) or Krebs et al. (Journal ofImmunological Methods, 254:67-84, 2001).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward et al., Nature 341:544-546, 1989; McCafferty et al.Nature, 348:552-554, 1990; Holt et al. Trends in Biotechnology 21,484-490, 2003), which consists of a VH or a VL domain; (v) isolated CDRregions; (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal. Science, 242, 423-426, 1988; Huston PNAS USA, 85, 5879-5883, 1988);(viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix)“diabodies”, multivalent or multispecific fragments constructed by genefusion (WO94/13804; Holliger et al, PNAS USA 90:6444-6448, 1993a). Fv,scFv or diabody molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al, NatureBiotech, 14:1239-1245, 1996). Minibodies comprising a scFv joined to aCH3 domain may also be made (Hu et al, Cancer Res., 56, 3055-3061,1996). Other examples of binding fragments arc Fab′, which differs fromFab fragments by the addition of a few residues at the carboxyl terminusof the heavy chain CH1 domain, including one or more cysteines from theantibody hinge region, and Fab′-SH, which is a Fab′ fragment in whichthe cysteine residue(s) of the constant domains bear a free thiol group.

Antibody fragments of the invention can be obtained starting from any ofthe antibody molecules described herein, e.g. antibody moleculescomprising VH and/or VL domains or CDRs of any of Antibodies 1 to 42, bymethods such as digestion by enzymes, such as pepsin or papain and/or bycleavage of the disulfide bridges by chemical reduction. In anothermanner, the antibody fragments comprised in the present invention can beobtained by techniques of genetic recombination likewise well known tothe person skilled in the art or else by peptide synthesis by means of,for example, automatic peptide synthesizers such as those supplied bythe company Applied Biosystems, etc., or by nucleic acid synthesis andexpression.

Functional antibody fragments according to the present invention includeany functional fragment whose half-life is increased by a chemicalmodification, especially by PEGylation, or by incorporation in aliposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt et al. Trends in Biotechnology 21, 484-490, 2003). VH dAbsoccur naturally in camelids (e.g. camel, llama) and may be produced byimmunizing a camelid with a target antigen, isolating antigen-specific Bcells and directly cloning dAb genes from individual B cells. dAbs arealso producible in cell culture. Their small size, good solubility andtemperature stability makes them particularly physiologically useful andsuitable for selection and affinity maturation. A binding member of thepresent invention may be a dAb comprising a VH or VL domainsubstantially as set out herein, or a VH or VL domain comprising a setof CDRs substantially as set out herein.

As used herein, the phrase “substantially as set out” refers to thecharacteristic(s) of the relevant CDRs of the VH or VL domain of bindingmembers described herein will be either identical or highly similar tothe specified regions of which the sequence is set out herein. Asdescribed herein, the phrase “highly similar” with respect to specifiedregion(s) of one or more variable domains, it is contemplated that from1 to about 12, e.g. from 1 to 8, including 1 to 7, 1 to 6, 1 to 5, 1 to4, 1 to 3, or 1 or 2, amino acid substitutions may be made in the CDRsof the VH and/or VL domain.

Antibodies of the invention include bispecific antibodies. Bispecific orbifunctional antibodies form a second generation of monoclonalantibodies in which two different variable regions are combined in thesame molecule (Holliger, P. & Winter, G. 1999 Cancer and metastasis rev.18:411-419, 1999). Their use has been demonstrated both in thediagnostic field and in the therapy field from their capacity to recruitnew effector functions or to target several molecules on the surface oftumor cells. Where bispecific antibodies are to be used, these may beconventional bispecific antibodies, which can be manufactured in avariety of ways (Holliger et al, PNAS USA 90:6444-6448, 1993), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. These antibodies can beobtained by chemical methods (Glennie et al., 1987 J. Immunol. 139,2367-2375; Repp et al., J. Hemat. 377-382, 1995) or somatic methods(Staerz U. D. and Bevan M. J. PNAS 83, 1986; et al., Method Enzymol.121:210-228, 1986) but likewise by genetic engineering techniques whichallow the heterodimerization to be forced and thus facilitate theprocess of purification of the antibody sought (Merchand et al. NatureBiotech, 16:677-681, 1998). Examples of bispecific antibodies includethose of the BiTE™ technology in which the binding domains of twoantibodies with different specificity can be used and directly linkedvia short flexible peptides. This combines two antibodies on a shortsingle polypeptide chain. Diabodies and scFv can be constructed withoutan Fc region, using only variable domains, potentially reducing theeffects of anti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against IL-4Rα, then a library can be made where the other armis varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by alternative engineeringmethods as described in Ridgeway et al, (Protein Eng., 9:616-621, 1996).

Various methods are available in the art for obtaining antibodiesagainst IL-4Rα. The antibodies may be monoclonal antibodies, especiallyof human, murine, chimeric or humanized origin, which can be obtainedaccording to the standard methods well known to the person skilled inthe art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988) or tothe technique of preparation from hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

Monoclonal antibodies can be obtained, for example, from an animal cellimmunized against IL-4Rα, or one of their fragments containing theepitope recognized by said monoclonal antibodies. The IL-4Rα, or one ofits fragments, can especially be produced according to the usual workingmethods, by genetic recombination starting with a nucleic acid sequencecontained in the cDNA sequence coding for IL-4Rα or fragment thereof, bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the IL-4Rα and/or fragment thereof. Themonoclonal antibodies can, for example, be purified on an affinitycolumn on which IL-4Rα or one of its fragments containing the epitoperecognized by said monoclonal antibodies, has previously beenimmobilized. More particularly, the monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itself,followed or not followed by exclusion chromatography on Sepharose gel inorder to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In one embodiment, the whole of thesetechniques can be used simultaneously or successively.

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. An antibody antigen-binding site may comprise anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

Isolated

This refers to the state in which binding members of the invention, ornucleic acid encoding such binding members, will generally be inaccordance with the present invention. Thus, binding members, includingVH and/or VL domains, and encoding nucleic acid molecules and vectorsaccording to the present invention may be provided isolated and/orpurified, e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or genes of origin other than the sequence encodinga polypeptide with the required function. Isolated members and isolatednucleic acid will be free or substantially free of material with whichthey are naturally associated such as other polypeptides or nucleicacids with which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC85110503)) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

Heterogeneous preparations comprising anti-IL-4Rα antibody moleculesalso form part of the invention. For example, such preparations may bemixtures of antibodies with full-length heavy chains and heavy chainslacking the C-terminal lysine, with various degrees of glycosylationand/or with derivatized amino acids, such as cyclization of anN-terminal glutamic acid to form a pyroglutamic acid residue.

As noted above, a binding member in accordance with the presentinvention modulates and may neutralise a biological activity of IL-4Rα.As described herein, IL-4Rα-binding members of the present invention maybe optimised for neutralizing potency. Generally, potency optimisationinvolves mutating the sequence of a selected binding member (normallythe variable domain sequence of an antibody) to generate a library ofbinding members, which are then assayed for potency and the more potentbinding members are selected. Thus selected “potency-optimised” bindingmembers tend to have a higher potency than the binding member from whichthe library was generated. Nevertheless, high potency binding membersmay also be obtained without optimisation, for example a high potencybinding member may be obtained directly from an initial screen e.g. abiochemical neutralization assay. A “potency optimized” binding memberrefers to a binding member with an optimized potency of binding orneutralization of a particular activity or downstream function ofIL-4Rα. Assays and potencies are described in more detail elsewhereherein. The present invention provides both potency-optimized andnon-optimized binding members, as well as methods for potencyoptimization from a selected binding member. The present invention thusallows the skilled person to generate binding members having highpotency.

Although potency optimization may be used to generate higher potencybinding members from a given binding member, it is also noted that highpotency binding members may be obtained even without potencyoptimization.

In a further aspect, the present invention provides a method ofobtaining one or more binding members able to bind the antigen, themethod including bringing into contact a library of binding membersaccording to the invention and said antigen, and selecting one or morebinding members of the library able to bind said antigen.

The library may be displayed on particles or molecular complexes, e.g.replicable genetic packages such as yeast, bacterial or bacteriophage(e.g. T7) particles, viruses, cells or covalent, ribosomal or other invitro display systems, each particle or molecular complex containingnucleic acid encoding the antibody VH variable domain displayed on it,and optionally also a displayed VL domain if present. Phage display isdescribed in WO92/01047 and e.g. U.S. Pat. No. 5,969,108, U.S. Pat. No.5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat. No. 5,858,657, U.S. Pat.No. 5,871,907, U.S. Pat. No. 5,872,215, U.S. Pat. No. 5,885,793, U.S.Pat. No. 5,962,255, U.S. Pat. No. 6,140,471, U.S. Pat. No. 6,172,197,U.S. Pat. No. 6,225,447, U.S. Pat. No. 6,291,650, U.S. Pat. No.6,492,160 and U.S. Pat. No. 6,521,404, each of which is hereinincorporated by reference in their entirety.

Following selection of binding members able to bind the antigen anddisplayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected binding member.Such nucleic acid may be used in subsequent production of a bindingmember or an antibody VH or VL variable domain by expression fromnucleic acid with the sequence of nucleic acid taken from abacteriophage or other particle or molecular complex displaying a saidselected binding member.

An antibody VH domain with the amino acid sequence of an antibody VHdomain of a said selected binding member may be provided in isolatedform, as may a binding member comprising such a VH domain.

Ability to bind IL-4Rα and/or ability to compete with e.g. a parentantibody molecule (e.g. Antibody 1) or an optimised antibody molecule,Antibodies 2 to 42 (e.g. in scFv format and/or IgG format, e.g. IgG 1,IgG2 or IgG4) for binding to IL-4Rα, may be further tested. Ability toneutralize IL-4Rα may be tested, as discussed further elsewhere herein.

A binding member according to the present invention may bind IL-4Rα withthe affinity of one of Antibodies 1 to 42, e.g. in scFv or IgG 1 or IgG2or IgG4 format, or with an affinity that is better.

A binding member according to the present invention may neutralize abiological activity of IL-4Rα with the potency of one of Antibodies 1 to42 e.g. in scFv or IgG 1 or IgG2 or IgG4 format, or with a potency thatis better.

Binding affinity and neutralization potency of different binding memberscan be compared under appropriate conditions.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. Following the lead of computational chemistryin applying multivariate data analysis techniques to thestructure/property-activity relationships (Wold et al. Multivariate dataanalysis in chemistry. Chemometrics—Mathematics and Statistics inChemistry (Ed.: B. Kowalski), D. Reidel Publishing Company, Dordrecht,Holland, 1984 (ISBN 90-277-1846-6)) quantitative activity-propertyrelationships of antibodies can be derived using well-known mathematicaltechniques such as statistical regression, pattern recognition andclassification (Norman et al. Applied Regression Analysis.Wiley-Interscience; 3^(rd) edition (April 1998) ISBN: 0471170828;Kandel, Abraham & Backer, Computer-Assisted Reasoning in ClusterAnalysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847;Principles of Multivariate Analysis: A User's Perspective (OxfordStatistical Science Series, No 22 (Paper)). Oxford University Press;(December 2000), ISBN: 0198507089; Witten & Frank Data Mining: PracticalMachine Learning Tools and Techniques with Java Implementations. MorganKaufmann; (Oct. 11, 1999), ISBN: 1558605525; Denison DGT. (Editor),Holmes, C C. et al. Bayesian Methods for Nonlinear Classification andRegression (Wiley Series in Probability and Statistics). John Wiley &Sons; (July 2002), ISBN: 0471490369; Ghose, A K. & Viswanadhan, V N.Combinatorial Library Design and Evaluation Principles, Software, Tools,and Applications in Drug Discovery. ISBN: 0-8247-0487-8). The propertiesof antibodies can be derived from empirical and theoretical models (forexample, analysis of likely contact residues or calculatedphysicochemical property) of antibody sequence, functional andthree-dimensional structures and these properties can be consideredsingly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis typically formed by six loops of polypeptide: three from the lightchain variable domain (VL) and three from the heavy chain variabledomain (VH). Analysis of antibodies of known atomic structure haselucidated relationships between the sequence and three-dimensionalstructure of antibody combining sites (Chothia et al. Journal MolecularBiology 1992227, 799-817, 1992; Al-Lazikani et al. Journal MolecularBiology 273(4):927-948, 1997). These relationships imply that, exceptfor the third region (loop) in VH domains, binding site loops have oneof a small number of main-chain conformations: canonical structures. Thecanonical structure formed in a particular loop has been shown to bedetermined by its size and the presence of certain residues at key sitesin both the loop and in framework regions (Chothia et al, JournalMolecular Biology 1992227, 799-817, 1992; Al-Lazikani supra).

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments. In astructural approach, a model can be created of the antibody molecule(Chothia et al. Science, 223:755-758, 1986) using any freely availableor commercial package such as WAM (Whitelegg & Rees, Prot. Eng.,12:815-824, 2000). A protein visualisation and analysis software packagesuch as Insight II (Accelerys, Inc.) or Deep View (Guex & Peitsch,Electrophoresis (1997) 18, 2714-2723) may then be used to evaluatepossible substitutions at each position in the CDR. This information maythen be used to make substitutions likely to have a minimal orbeneficial effect on activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and binding membersgenerally are available in the art. Variant sequences may be made, withsubstitutions that may or may not be predicted to have a minimal orbeneficial effect on activity, and tested for ability to bind and/orneutralize IL-4Rα and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 12 alterations, less than about 10alterations, or less than about 6 alterations, maybe 5, 4, 3, 2 or 1.Alterations may be made in one or more framework regions and/or one ormore CDRs. The alterations normally do not result in loss of function,so a binding member comprising a thus-altered amino acid sequence mayretain an ability to bind and/or neutralize IL-4Rα. For example, it mayretain the same quantitative binding and/or neutralizing ability as abinding member in which the alteration is not made, e.g. as measured inan assay described herein. The binding member comprising a thus-alteredamino acid sequence may have an improved ability to bind and/orneutralize IL-4Rα. Indeed, Antibodies 21 to 42, generated from randommutagenesis of Antibody 20, exhibits substitutions relative to Antibody20, mostly within the various framework regions and each of these stillbind and/or neutralises IL-4Rα, indeed some show improved ability tobind and/or neutralize IL-4Rα.

Alteration may comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Example numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the 20 “standard”L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C,K, R, H, D, E by their standard single-letter codes. Non-standard aminoacids include any other residue that may be incorporated into apolypeptide backbone or result from modification of an existing aminoacid residue. Non-standard amino acids may be naturally occurring ornon-naturally occurring. Several naturally occurring non-standard aminoacids arc known in the art, such as 4-hydroxyproline, 5-hydroxylysine,3-methylhistidine, N-acetylserine, etc. (Voet & Voet, Biochemistry, 2ndEdition, (Wiley) 1995). Those amino acid residues that are derivatisedat their N-alpha position will only be located at the N-terminus of anamino-acid sequence. Normally in the present invention an amino acid isan L-amino acid, but in some embodiments it may be a D-amino acid.Alteration may therefore comprise modifying an L-amino acid into, orreplacing it with, a D-amino acid. Methylated, acetylated and/orphosphorylated forms of amino acids are also known, and amino acids inthe present invention may be subject to such modification.

Amino acid sequences in antibody domains and binding members of theinvention may comprise non-natural or non-standard amino acids describedabove. In some embodiments non-standard amino acids (e.g. D-amino acids)may be incorporated into an amino acid sequence during synthesis, whilein other embodiments the non-standard amino acids may be introduced bymodification or replacement of the “original” standard amino acids aftersynthesis of the amino acid sequence.

Use of non-standard and/or non-naturally occurring amino acids increasesstructural and functional diversity, and can thus increase the potentialfor achieving desired IL-4Rα-binding and neutralizing properties in abinding member of the invention. Additionally, D-amino acids andanalogues have been shown to have better pharmacokinetic profilescompared with standard L-amino acids, owing to in vivo degradation ofpolypeptides having L-amino acids after administration to an animal e.g.a human.

Novel VH or VL regions carrying CDR-derived sequences of the inventionmay be generated using random mutagenesis of one or more selected VHand/or VL genes to generate mutations within the entire variable domain.Such a technique is described by Gram et al. (Proc. Natl. Acad. Sci.,USA, 89:3576-3580, 1992), who used error-prone PCR. In some embodimentsone or two amino acid substitutions are made within an entire variabledomain or set of CDRs. Another method that may be used is to directmutagenesis to CDR regions of VH or VL genes. Such techniques aredisclosed by Barbas et al. (Proc. Natl. Acad. Sci., 91:3809-3813, 1994)and Schier et al. (J. Mol. Biol. 263:551-567, 1996).

All the above-described techniques are known as such in the art and theskilled person will be able to use such techniques to provide bindingmembers of the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining anantibody antigen-binding site for IL-4Rα, the method comprisingproviding by way of addition, deletion, substitution or insertion of oneor more amino acids in the amino acid sequence of a VH domain set outherein a VH domain which is an amino acid sequence variant of the VHdomain, optionally combining the VH domain thus provided with one ormore VL domains, and testing the VH domain or VH/VL combination orcombinations to identify a binding member or an antibody antigen-bindingsite for IL-4Rα and optionally with one or more functional properties,e.g. ability to neutralize IL-4Rα activity. Said VL domain may have anamino acid sequence which is substantially as set out herein. Ananalogous method may be employed in which one or more sequence variantsof a VL domain disclosed herein are combined with one or more VHdomains.

As noted above, a CDR amino acid sequence substantially as set outherein may be carried as a CDR in a human antibody variable domain or asubstantial portion thereof. The HCDR3 sequences substantially as setout herein represent embodiments of the present invention and forexample each of these may be carried as a HCDR3 in a human heavy chainvariable domain or a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A variable domain can be derived from anon-human antibody. A CDR sequence of the invention (e.g. CDR3) may beintroduced into a repertoire of variable domains lacking a CDR (e.g.CDR3), using recombinant DNA technology. For example, Marks et al.(Bio/Technology, 10:779-783, 1992) describe methods of producingrepertoires of antibody variable domains in which consensus primersdirected at or adjacent to the 5′ end of the variable domain area areused in conjunction with consensus primers to the third framework regionof human VH genes to provide a repertoire of VH variable domains lackinga CDR3. Marks et al. further describe how this repertoire may becombined with a CDR3 of a particular antibody. Using analogoustechniques, the CDR3-derived sequences of the present invention may beshuffled with repertoires of VH or VL domains lacking a CDR3, and theshuffled complete VH or VL domains combined with a cognate VL or VHdomain to provide binding members of the invention. The repertoire maythen be displayed in a suitable host system such as the phage displaysystem of WO92/01047, which is herein incorporated by reference in itsentirety, or any of a subsequent large body of literature, includingKay, Winter & McCafferty (Phage Display of Peptides and Proteins: ALaboratory Manual, San Diego: Academic Press, 1996), so that suitablebinding members may be selected. A repertoire may consist of fromanything from 10⁴ individual members upwards, for example at least 10⁵,at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least 10¹⁰members. Other suitable host systems include, but are not limited to,yeast display, bacterial display, T7 display, viral display, celldisplay, ribosome display and covalent display.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 370:389-391, 1994), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A method of preparing a binding member for IL-4Rα antigen is provided,which method comprises:

(a) providing a starting repertoire of nucleic acids encoding a VHdomain which either include a CDR3 to be replaced or lack a CDR3encoding region;

(b) combining said repertoire with a donor nucleic acid encoding anamino acid sequence substantially as set out herein for a VH CDR3 suchthat said donor nucleic acid is inserted into the CDR3 region in therepertoire, so as to provide a product repertoire of nucleic acidsencoding a VH domain;

(c) expressing the nucleic acids of said product repertoire;

(d) selecting a binding member for IL-4Rα; and

(e) recovering said binding member or nucleic acid encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain that either include a CDR3 to be replaced or lack a CDR3 encodingregion.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for IL-4Rα.

Alternatively, nucleic acid encoding the VH and/or VL domains of any ofa binding member of the present invention, e.g. Antibodies 1-42, can besubjected to mutagenesis (e.g. targeted or random) to generate one ormore mutant nucleic acids. Binding members encoded by these sequencescan then be generated.

In one embodiment, one or more of Antibodies 1 to 42 HCDR1, HCDR2 andHCDR3, or an Antibody 1 to 42 set of HCDRs, may be employed, and/or oneor more of Antibodies 1 to 42 LCDR1, LCDR2 and LCDR3 or an Antibody 1 to42 set of LCDRs may be employed.

According to one aspect of the invention there is provided a method forproducing a binding member that binds IL-4Rα, which method comprises:

providing starting nucleic acid encoding a VH domain or a VL domain, ora starting repertoire of nucleic acids each encoding a VH or VL domain,wherein the VH or VL domains either comprise a CDR1, CDR2 and/or CDR3 tobe replaced or lack a CDR1, CDR2 and/or CDR3 encoding region;

combining said starting nucleic acid or starting repertoire with donornucleic acid or donor nucleic acids encoding or produced by mutation ofthe amino acid sequence of an CDR1, CDR2, and/or CDR3 of any ofAntibodies 1-42, such that said donor nucleic acid is or donor nucleicacids are inserted into the CDR1, CDR2 and/or CDR3 region in thestarting nucleic acid or starting repertoire, so as to provide a productrepertoire of nucleic acids encoding VH or VL domains;

expressing the nucleic acids of said product repertoire to produceproduct VH or VL domains;

optionally combining said product VH or VL domains with one or morecompanion VL or VH domains;

selecting a binding member for IL-4Rα, which binding member comprises aproduct VH or VL domain and optionally a companion VL or VH domain; andrecovering said binding member or nucleic acid encoding it.

In particular embodiments the donor nucleic acid is produced by targetedor random mutagenesis of the VH or VL domains or any CDR region therein.

In another embodiment, the product VH or VL domain is attached to anantibody constant region.

In another embodiment the product VH or VL domain and a companion VL orVH domain respectively, is comprised in an IgG, scFV or Fab antibodymolecule.

In another embodiment the recovered binding member or antibody moleculeis tested for ability to neutralise IL-4Rα.

In another embodiment the antibody molecule is formulated into acomposition comprising at least one additional component. Such componentcould, for example, be an inert pharmaceutical excipient or carrier.

In some embodiments, a substantial portion of an immunoglobulin variabledomain will comprise at least the three CDR regions, together with theirintervening framework regions. The portion may also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of binding members ofthe present invention made by recombinant DNA techniques may result inthe introduction of N- or C-terminal residues encoded by linkersintroduced to facilitate cloning or other manipulation steps. Othermanipulation steps include the introduction of linkers to join variabledomains of the invention to further protein sequences including antibodyconstant regions, other variable domains (for example in the productionof diabodies) or detectable/functional labels as discussed in moredetail elsewhere herein.

Although in some aspects of the invention, binding members comprise apair of VH and VL domains, single binding domains based on either VH orVL domain sequences form further aspects of the invention. It is knownthat single immunoglobulin domains, especially VH domains, are capableof binding target antigens in a specific manner. For example, see thediscussion of dAbs above.

In the case of either of the single binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain binding member able to bind IL-4Rα. This may be achieved byphage display screening methods using the so-called hierarchical dualcombinatorial approach as disclosed in WO92/01047, herein incorporatedby reference in its entirety, in which an individual colony containingeither an H or L chain clone is used to infect a complete library ofclones encoding the other chain (L or H) and the resulting two-chainbinding member is selected in accordance with phage display techniquessuch as those described in that reference. This technique is alsodisclosed in Marks et al. (Bio/Technology, 10:779-783, 1992).

Binding members of the present invention may further comprise antibodyconstant regions or parts thereof, e.g. human antibody constant regionsor parts thereof. For example, a VL domain may be attached at itsC-terminal end to antibody light chain constant domains including humanCκ or Cλ chains, e.g. Cλ chains. Similarly, a binding member based on aVH domain may be attached at its C-terminal end to all or part (e.g. aCH1 domain) of an immunoglobulin heavy chain derived from any antibodyisotype, e.g. IgG, IgA, IgD, IgY, IgE and IgM and any of the isotypesub-classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2; particularlyIgG1 and IgG4). IgG1 is advantageous, due to its effector function andease of manufacture. Any synthetic or other constant region variant thathas these properties and stabilizes variable regions is also useful inembodiments of the present invention.

The term “isotype” refers to the classification of an antibody's heavyor light chain constant region. The constant domains of antibodies arenot involved in binding to antigen, but exhibit various effectorfunctions. Depending on the amino acid sequence of the heavy chainconstant region, a given human antibody or immunoglobulin can beassigned to one of five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM. Several of these classes may be further divided intosubclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma3), and IgG4 (gamma 4), and IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1 and IgG3 are known to mediate ADCC in humans. Human light chainconstant regions may be classified into two major classes, kappa andlambda.

Antibody Format

The present invention also includes binding members of the invention,and in particular the antibodies of the invention, that have modifiedIgG constant domains. Antibodies of the human 1gG class, which havefunctional characteristics such a long half-life in serum and theability to mediate various effector functions are used in certainembodiments of the invention (Monoclonal Antibodies: Principles andApplications, Wiley-Liss, Inc., Chapter 1 (1995)). The human IgG classantibody is further classified into the following 4 subclasses: IgG1,IgG2, IgG3 and IgG4. A large number of studies have so far beenconducted for ADCC and CDC as effector functions of the IgG classantibody, and it has been reported that among antibodies of the humanIgG class, the IgG1 subclass has the highest ADCC activity and CDCactivity in humans (Chemical Immunology, 65, 88 (1997)).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells (e.g.,Natural Killer (NK) cells, neutrophils, and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In one embodiment, such cells are human cells. While not wishingto be limited to any particular mechanism of action, these cytotoxiccells that mediate ADCC generally express Fc receptors (FcRs). Theprimary cells for mediating ADCC, NK cells, express FcγRIII, whereasmonocytes express FcγRI, FcγRII, FcγRIII and/or FcγRIV. FcR expressionon hematopoietic cells is summarized in Ravetch and Kinet, Annu Rev.Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an invitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 orU.S. Pat. No. 5,821,337 may be performed. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecules of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA),95:652-656 (1998).

Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to initiate complement activation and lyse a target in thepresence of complement. The complement activation pathway is initiatedby the binding of the first component of the complement system (Clq) toa molecule (e.g., an antibody) complexed with a cognate antigen. Toassess complement activation, a CDC assay, e.g., as described inGazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

Expression of ADCC activity and CDC activity of the human IgG1 subclassantibodies generally involves binding of the Fc region of the antibodyto a receptor for an antibody (hereinafter referred to as “FcγR”)existing on the surface of effector cells such as killer cells, naturalkiller cells or activated macrophages. Various complement components canbe bound. Regarding the binding, it has been suggested that severalamino acid residues in the hinge region and the second domain of Cregion (hereinafter referred to as “Cγ2 domain”) of the antibody areimportant (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319(1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain inthe Cγ2 domain (Chemical Immunology, 65, 88 (1997)) is also important.

“Effector cells” are leukocytes that express one or more FcRs andperform effector functions. The cells express at least FcγRI, FCγRII,FcγRIII and/or FcγRIV and carry out ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, the FcR is anative sequence human FcR. Moreover, in certain embodiments, the FcR isone that binds an IgG antibody (a gamma receptor) and includes receptorsof the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelicvariants and alternatively spliced forms of these receptors. FcγRIIreceptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an“inhibiting receptor”), which have similar amino acid sequences thatdiffer primarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See, Daëron, Annu Rev. Immunol., 15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu Rev. Immunol.,9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol.,24:249 (1994)).

Anti-IL-4Rα antibodies can be modified with respect to effectorfunction, e.g., so as to enhance ADCC and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in the Fc region of an antibody.Cysteine residue(s) may also be introduced in the Fc region, allowingfor interchain disulfide bond formation in this region. In this way ahomodimeric antibody can be generated that may have improvedinternalization capability and or increased complement-mediated cellkilling and ADCC (Caron et al., J. Exp. Med., 176:1191-1195 (1992) andShopes, J. Immunol., 148:2918-2922 (1992)). Heterobifunctionalcross-linkers can also be used to generate homodimeric antibodies withenhanced anti-tumor activity (Wolff et al., Cancer Research,53:2560-2565 (1993)). Antibodies can also be engineered to have two ormore Fc regions resulting in enhanced complement lysis and ADCCcapabilities (Stevenson et al., Anti-Cancer Drug Design, (3): 219-230(1989)).

Other methods of engineering Fc regions of antibodies so as to altereffector functions are known in the art (e.g., U.S. Patent PublicationNo. 20040185045 and PCT Publication No. WO 2004/016750, both to Koeniget al., which describe altering the Fc region to enhance the bindingaffinity for FcγRIIB as compared with the binding affinity for FCγRIIA;see also PCT Publication Nos. WO 99/58572 to Armour et al., WO 99/51642to Idusogie et al., and U.S. Pat. No. 6,395,272 to Deo et al.; thedisclosures of which are incorporated herein in their entireties).Methods of modifying the Fc region to decrease binding affinity toFcγRIIB are also known in the art (e.g., U.S. Patent Publication No.20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.,the disclosures of which are incorporated herein in their entireties).Modified antibodies having variant Fc regions with enhanced bindingaffinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fcregion have also been described (e.g., PCT Publication No. WO2004/063351, to Stavenhagen et al.; the disclosure of which isincorporated herein in its entirety).

At least four different types of FcγR have been found, which arerespectively called FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIV. In human, FcγRII and FcγRI11 are further classified into FcγRIIaand FcγRIIb, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membraneprotein belonging to the immunoglobulin superfamily, FcγRII, FcγRIII,and FcγRIV have an a chain having an extracellular region containing twoimmunoglobulin-like domains, FcγRI has an a chain having anextracellular region containing three immunoglobulin-like domains, as aconstituting component, and the α chain is involved in the IgG bindingactivity. In addition, FcγRI and FcγRIII have a γ chain or ζ chain as aconstituting component which has a signal transduction function inassociation with the a chain (Annu. Rev. Immunol., 18, 709 (2000), Annu.Rev. Immunol., 19, 275 (2001)). FcγRIV has been described by Bruhns etal., Clin. Invest. Med., (Canada) 27:3 D (2004).

To assess ADCC activity of an anti-IL-4Rα antibody of interest, an invitro ADCC assay can be used, such as that described in U.S. Pat. No.5,500,362 or U.S. Pat. No. 5,821,337. The assay may also be performedusing a commercially available kit, e.g. CytoTox 96® (Promega). Usefuleffector cells for such assays include, but are not limited toperipheral blood mononuclear cells (PBMC), Natural Killer (NK) cells,and NK cell lines. NK cell lines expressing a transgenic Fc receptor(e.g. CD16) and associated signaling polypeptide (e.g. FCεRI-γ) may alsoserve as effector cells (see, e.g. WO 2006/023148 A2 to Campbell). Forexample, the ability of any particular antibody to mediate lysis of thetarget cell by complement activation and/or ADCC can be assayed. Thecells of interest are grown and labeled in vitro; the antibody is addedto the cell culture in combination with immune cells which may beactivated by the antigen antibody complexes; i.e., effector cellsinvolved in the ADCC response. The antibody can also be tested forcomplement activation. In either case, cytolysis of the target cells isdetected by the release of label from the lysed cells. The extent oftarget cell lysis may also be determined by detecting the release ofcytoplasmic proteins (e.g. LDH) into the supernatant. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibodies that are capable ofmediating human ADCC in the in vitro test can then be usedtherapeutically in that particular patient. ADCC activity of themolecule of interest may also be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci.(USA) 95:652-656 (1998). Moreover, techniques for modulating (i.e.,increasing or decreasing) the level of ADCC, and optionally CDCactivity, of an antibody are well-known in the art. See, e.g., U.S. Pat.No. 6,194,551. Antibodies of the present invention may be capable or mayhave been modified to have the ability of inducing ADCC and/or CDC.Assays to determine ADCC function can be practiced using human effectorcells to assess human ADCC function. Such assays may also include thoseintended to screen for antibodies that induce, mediate, enhance, blockcell death by necrotic and/or apoptotic mechanisms. Such methodsincluding assays utilizing viable dyes, methods of detecting andanalyzing caspases, and assays measuring DNA breaks can be used toassess the apoptotic activity of cells cultured in vitro with ananti-IL-4Rα antibody of interest.

For example, Annexin V or TdT-mediated dUTP nick-end labeling (TUNEL)assays can be carried out as described in Decker et al., Blood (USA)103:2718-2725 (2004) to detect apoptotic activity. The TUNEL assayinvolves culturing the cell of interest with fluorescein-labeled dUTPfor incorporation into DNA strand breaks. The cells are then processedfor analysis by flow cytometry. The Annexin V assay detects theappearance of phosphatidylserine (PS) on the outside of the plasmamembrane of apoptotic cells using a fluorescein-conjugated Annexin Vthat specifically recognizes the exposed PS molecules. Concurrently, aviable dye such as propidium iodide can be used to exclude lateapoptotic cells. The cells are stained with the labeled Annexin V andare analyzed by flow cytometry.

Thus according to a further aspect of the invention there is providedbinding members, in particular antibodies, which have been modified soas to change, i.e. increase, decrease or eliminate, the biologicaleffector function of the binding members, for example antibodies withmodified Fc regions. In some embodiments, the binding members orantibodies as disclosed herein can be modified to enhance theircapability of fixing complement and participating incomplement-dependent cytotoxicity (CDC). In other embodiments, thebinding members or antibodies can be modified to enhance theircapability of activating effector cells and participating inantibody-dependent cytotoxicity (ADCC). In yet other embodiments, thebinding members or antibodies as disclosed herein can be modified bothto enhance their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC) and to enhancetheir capability of fixing complement and participating incomplement-dependent cytotoxicity (CDC).

In some embodiments, the binding members or antibodies as disclosedherein can be modified to reduce their capability of fixing complementand participating in complement-dependent cytotoxicity (CDC). In otherembodiments, the binding members or antibodies can be modified to reducetheir capability of activating effector cells and participating inantibody-dependent cytotoxicity (ADCC). In yet other embodiments, thebinding members or antibodies as disclosed herein can be modified bothto reduce their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC) and to reducetheir capability of fixing complement and participating incomplement-dependent cytotoxicity (CDC).

In one embodiment, a binding member with an Fc variant region hasenhanced ADCC activity relative to a comparable molecule. In a specificembodiment, a binding member with an Fc variant region has ADCC activitythat is at least 2 fold, or at least 3 fold, or at least 5 fold or atleast 10 fold or at least 50 fold or at least 100 fold greater than thatof a comparable molecule. In another specific embodiment, a bindingmember with an Fc variant region has enhanced binding to the Fc receptorFcγRIIIA and has enhanced ADCC activity relative to a comparablemolecule. In other embodiments, the binding member with an Fc variantregion has both enhanced ADCC activity and enhanced serum half-liferelative to a comparable molecule.

In one embodiment, a binding member with an Fc variant region hasreduced ADCC activity relative to a comparable molecule. In a specificembodiment, a binding member with an Fc variant region has ADCC activitythat is at least 2 fold, or at least 3 fold, or at least 5 fold or atleast 10 fold or at least 50 fold or at least 100 fold lower than thatof a comparable molecule. In another specific embodiment, the bindingmember with an Fc variant region has reduced binding to the Fc receptorFcγRIIIA and has reduced ADCC activity relative to a comparablemolecule. In other embodiments, the binding member with an Fc variantregion has both reduced ADCC activity and enhanced serum half-liferelative to a comparable molecule.

In one embodiment, the binding member with an Fc variant region hasenhanced CDC activity relative to a comparable molecule. In a specificembodiment the binding member with an Fc variant region has CDC activitythat is at least 2 fold, or at least 3 fold, or at least 5 fold or atleast 10 fold or at least 50 fold or at least 100 fold greater than thatof a comparable molecule. In other embodiments, the binding member withan Fc variant region has both enhanced CDC activity and enhanced serumhalf-life relative to a comparable molecule.

In one embodiment, the binding member with an Fc variant region hasreduced binding to one or more Fc ligand relative to a comparablemolecule. In another embodiment, the binding member with an Fc variantregion has an affinity for an Fc ligand that is at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200 foldlower than that of a comparable molecule. In a specific embodiment, thebinding member with an Fc variant region has reduced binding to an Fcreceptor. In another specific embodiment, the binding member with an Fcvariant region has reduced binding to the Fc receptor FcγRIIIA. In afurther specific embodiment, an binding member with an Fc variant regiondescribed herein has an affinity for the Fc receptor FcγRIIIA that is atleast about 5 fold lower than that of a comparable molecule, whereinsaid Fc variant has an affinity for the Fc receptor FcγRIIB that iswithin about 2 fold of that of a comparable molecule. In still anotherspecific embodiment, the binding member with an Fc variant region hasreduced binding to the Fc receptor FcRn. In yet another specificembodiment, the binding member with an Fc variant region has reducedbinding to Clq relative to a comparable molecule.

In one embodiment, the binding member with the Fc variant region hasenhanced binding to one or more Fc ligand(s) relative to a comparablemolecule. In another embodiment, the binding member with the Fc variantregion has an affinity for an Fc ligand that is at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200 foldgreater than that of a comparable molecule. In a specific embodiment,the binding member with the Fc variant region has enhanced binding to anFc receptor. In another specific embodiment, the binding member with theFc variant region has enhanced binding to the Fc receptor FcγRIIIA. In afurther specific embodiment, the binding member with the Fc variantregion has enhanced biding to the Fc receptor FcγRIIB. In still anotherspecific embodiment, the binding member with the Fc variant region hasenhanced binding to the Fc receptor FcRn. In yet another specificembodiment, the binding member with the Fc variant region has enhancedbinding to Clq relative to a comparable molecule.

In one embodiment, an anti-IL-4Rα antibody of the invention comprises avariant Fc domain wherein said variant Fc domain has enhanced bindingaffinity to Fc gamma receptor JIB relative to a comparable non-variantFc domain. In a further embodiment, an anti-IL-4Rα antibody of theinvention comprises a variant Fc domain wherein said variant Fc domainhas an affinity for Fc gamma receptor JIB that is at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200 foldgreater than that of a comparable non-variant Fc domain.

In one embodiment, the present invention provides a binding member withan Fc variant region or formulations comprising these, wherein the Fcregion comprises a non-native amino acid residue at one or morepositions selected from the group consisting of 228, 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262,263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298,299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non-native amino acid residue at additional and/oralternative positions known to one skilled in the art (see, e.g., U.S.Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO05/092925 and WO 06/020114).

By “non-native amino acid residue”, we mean an amino acid residue thatis not present at the recited position in the naturally occurringprotein. Typically, this will mean that the or a native/natural aminoacid residue has been substituted for one or more other residues, whichmay comprise one of the other 20 naturally-occurring (common) aminoacids or a non-classical amino acids or a chemical amino acid analog.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general.

In a specific embodiment, the present invention provides a bindingmember with an variant Fc region or a formulation comprising suchbinding member with an variant Fc region, wherein the Fc regioncomprises at least one non-native amino acid residue selected from thegroup consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I,234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H,235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H,239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L,256E, 256M, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 264I,264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F,265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E,269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D,296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I,298T, 298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F,316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W,327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T,328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V,3301, 330F, 330R,330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q,331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R,331N, 331D, 331T, 332D,332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E,370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered bythe EU index as set forth in Kabat. Optionally, the Fc region maycomprise additional and/or alternative non-native amino acid residuesknown to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919;WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

It will be understood that Fc region as used herein includes thepolypeptides comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.). The“EU index as set forth in Kabat” refers to the residue numbering of thehuman IgG1 EU antibody as described in Kabat et al. supra. Fc may referto this region in isolation, or this region in the context of anantibody, antibody fragment, or Fc fusion protein. An variant Fc proteinmay be an antibody, Fc fusion, or any protein or protein domain thatcomprises an Fc region including, but not limited to, proteinscomprising variant Fc regions, which are non naturally occurringvariants of an Fc.

The present invention encompasses binding members with variant Fcregions, which have altered binding properties for an Fc ligand (e.g.,an Fc receptor, Clq) relative to a comparable molecule (e.g., a proteinhaving the same amino acid sequence except having a wild type Fcregion). Examples of binding properties include but are not limited to,binding specificity, equilibrium dissociation constant (K_(D)),dissociation and association rates (k_(off) and k_(on) respectively),binding affinity and/or avidity. It is generally understood that abinding molecule (e.g., a variant Fc protein such as an antibody) with alow K_(D) may be preferable to a binding molecule with a high K_(D).However, in some instances the value of the k_(on) or k_(off) may bemore relevant than the value of the K_(D). One skilled in the art candetermine which kinetic parameter is most important for a given antibodyapplication.

The affinities and binding properties of an Fc domain for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions. The serum half-life ofproteins comprising Fc regions may be increased by increasing thebinding affinity of the Fc region for FcRn. In one embodiment, the Fcvariant protein has enhanced serum half-life relative to comparablemolecule.

The term “antibody half-life” as used herein means a pharmacokineticproperty of an antibody that is a measure of the mean survival time ofantibody molecules following their administration. Antibody half-lifecan be expressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body or a specificcompartment thereof, for example, as measured in serum or plasma, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

In certain embodiments, the half-life of an anti-IL-4Rα antibody orcompositions and methods of the invention is at least about 4 to 7 days.In certain embodiments, the mean half-life of an anti-IL-4Rα antibody ofcompositions and methods of the invention is at least about 2 to 5 days,3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to11 days, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to18, 15 to 19, or 16 to 20 days. In other embodiments, the mean half-lifeof an anti-IL-4Rα antibody of compositions and methods of the inventionis at least about 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24days, 21 to 25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to29 days, or 26 to 30 days. In still further embodiments the half-life ofan anti-IL-4Rα antibody of compositions and methods of the invention canbe up to about 50 days. In certain embodiments, the half-lives ofantibodies of compositions and methods of the invention can be prolongedby methods known in the art. Such prolongation can in turn reduce theamount and/or frequency of dosing of the antibody compositions.Antibodies with improved in vivo half-lives and methods for preparingthem are disclosed in U.S. Pat. No. 6,277,375, U.S. Pat. No. 7,083,784;and International Publication Nos. WO 98/23289 and WO 97/3461.

The serum circulation of anti-IL-4Rα antibodies in vivo may also beprolonged by attaching inert polymer molecules such as high molecularweight polyethyleneglycol (PEG) to the antibodies with or without amultifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of the antibodies or via epsilon-aminogroups present on lysyl residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by size-exclusion or by ion-exchange chromatography.PEG-derivatized antibodies can be tested for binding activity as well asfor in vivo efficacy using methods known to those of skill in the art,for example, by immunoassays described herein.

Further, the antibodies of compositions and methods of the invention canbe conjugated to albumin in order to make the antibody more stable invivo or have a longer half-life in vivo. The techniques are well knownin the art, see, e.g., International Publication Nos. WO 93/15199, WO93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all ofwhich are incorporated herein by reference.

In certain embodiments, the half-life of a binding member or antibody asdisclosed herein and of compositions of the invention is at least about4 to 7 days. In certain embodiments, the mean half-life of a bindingmember or antibody as disclosed herein and of compositions of theinvention is at least about 2 to 5 days, 3 to 6 days, 4 to 7 days, 5 to8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days, 8 to 12, 9 to 13, 10 to14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 days.In other embodiments, the mean half-life of a binding member or antibodyas disclosed herein and of compositions of the invention is at leastabout 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24 days, 21 to25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to 29 days, or26 to 30 days. In still further embodiments the half-life of a bindingmember or antibody as disclosed herein and of compositions of theinvention can be up to about 50 days. In certain embodiments, thehalf-lives of antibodies and of compositions of the invention can beprolonged by methods known in the art. Such prolongation can in turnreduce the amount and/or frequency of dosing of the antibodycompositions. Antibodies with improved in vivo half-lives and methodsfor preparing them are disclosed in U.S. Pat. No. 6,277,375; U.S. Pat.No. 7,083,784; and International Publication Nos. WO 1998/23289 and WO1997/34361.

In another embodiment, the present invention provides a binding member,particularly an antibody with a variant Fc region, or a formulationcomprising these, wherein the Fc region comprises at least onenon-native modification at one or more positions selected from the groupconsisting of 239, 330 and 332, as numbered by the EU index as set forthin Kabat. In a specific embodiment, the present invention provides an Fcvariant, wherein the Fc region comprises at least one non-native aminoacid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may further comprise additional non-native amino acid at one ormore positions selected from the group consisting of 252, 254, and 256,as numbered by the EU index as set forth in Kabat.

In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non-native amino acidselected from the group consisting of 239D, 330L and 332E, as numberedby the EU index as set forth in Kabat and at least one normative aminoacid at one or more positions selected from the group consisting of252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

In another embodiment, the present invention provides a binding member,particularly an antibody with a variant Fc region, or a formulationcomprising these, wherein the Fc region comprises at least onenon-native amino acid at one or more positions selected from the groupconsisting of 234, 235 and 331, as numbered by the EU index as set forthin Kabat. In a specific embodiment, the present invention provides an Fcvariant, wherein the Fc region comprises at least one non-native aminoacid selected from the group consisting of 234F, 235F, 235Y, and 331S,as numbered by the EU index as set forth in Kabat. In a further specificembodiment, an Fc variant of the invention comprises the 234F, 235F, and331S amino acid residues, as numbered by the EU index as set forth inKabat. In another specific embodiment, an Fc variant of the inventioncomprises the 234F, 235Y, and 331S amino acid residues, as numbered bythe EU index as set forth in Kabat. Optionally, the Fc region mayfurther comprise additional non-native amino acid residues at one ormore positions selected from the group consisting of 252, 254, and 256,as numbered by the EU index as set forth in Kabat. In a specificembodiment, the present invention provides an Fc variant, wherein the Fcregion comprises at least one non-native amino acid selected from thegroup consisting of 234F, 235F, 235Y, and 331S, as numbered by the EUindex as set forth in Kabat; and at least one non-native amino acid atone or more positions selected from the group consisting of 252Y, 254Tand 256E, as numbered by the EU index as set forth in Kabat.

In a particular embodiment, the invention provides a binding member ofthe present invention with a variant Fc region, wherein the variantcomprises a tyrosine (Y) residue at position 252, a threonine (T)residue at position 254 and a glutamic acid (E) residue at position 256,as numbered by the EU index as set forth in Kabat.

The M252Y, S254T and T256E mutations, as numbered by the EU index as setforth in Kabat, hereinafter referred to as YTE mutations, have beenreported to increase serum half-life of a particular IgG1 antibodymolecule (Dall'Acqua et al. J. Biol. Chem. 281(33):23514-23524, 2006).

In a further embodiment, the invention provides a binding member of thepresent invention with a variant Fc region, wherein the variantcomprises a tyrosine (Y) residue at position 252, a threonine (T)residue at position 254, a glutamic acid (E) residue at position 256 anda proline (P) residue at position 241, as numbered by the EU index asset forth in Kabat.

The serine228proline mutation (S228P), as numbered by the EU index asset forth in Kabat, hereinafter referred to as the P mutation, has beenreported to increase the stability of a particular IgG4 molecule (Lu etal., J Pharmaceutical Sciences 97(2):960-969, 2008). Note: In Lu et al.it is referred to as position 241 because therein they use the Kabatnumbering system, not the “EU index” as set forth in Kabat.

This P mutation may be combined with L235E to further knock out ADCC.This combination of mutations is hereinafter referred to as the doublemutation (DM).

In a particular embodiment, the invention provides a binding member ofthe present invention with a variant Fc region, wherein the variantcomprises a phenylalanine (F) residue at position 234, a phenylalanine(F) residue or a glutamic acid (E) residue at position 235 and a serine(S) residue at position 331, as numbered by the EU index as set forth inKabat. Such a mutation combinations are hereinafter referred to as thetriple mutant (TM).

According to a further embodiment, the invention provides an antibody ofthe present invention in IgG1 format with the YTE mutations in the Fcregion.

According to a further embodiment, the invention provides an antibody ofthe present invention in IgG1 format with the TM mutations in the Fcregion.

According to a further embodiment, the invention provides an antibody ofthe present invention in IgG1 format with the YTE mutations and the TMmutations in the Fc region.

According to embodiment, the invention provides an antibody of thepresent invention in IgG4 format with the YTE and P mutations in the Fcregion.

According to embodiment, the invention provides an antibody of thepresent invention in IgG4 format with the YTE and DM mutations in the Fcregion.

According to particular embodiments of the inventions there is providedan antibody of the present invention in a format selected from: IgG1YTE, IgG1TM, IgG1 TM+YTE, IgG4 P, IgG4 DM, IgG4 YTE, IgG4 P+YTE and IgG4DM+YTE.

In terms of the nomenclature used, it will be appreciated that DM+YTEmeans that the constant domain Fc region possesses both the doublemutations (S228P and L235E) and the YTE mutations (M252Y, S254T andT256E).

Methods for generating non naturally occurring Fc regions are known inthe art. For example, amino acid substitutions and/or deletions can begenerated by mutagenesis methods, including, but not limited to,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA82:488-492, 1985), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guideto Methods and Applications”, Academic Press, San Diego, pp. 177-183,1990), and cassette mutagenesis (Wells et al., Gene 34:315-323, 1985).Preferably, site-directed mutagenesis is performed by theoverlap-extension PCR method (Higuchi, in “PCR Technology: Principlesand Applications for DNA Amplification”, Stockton Press, New York, pp.61-70, 1989). The technique of overlap-extension PCR (Higuchi, ibid.)can also be used to introduce any desired mutation(s) into a targetsequence (the starting DNA). For example, the first round of PCR in theoverlap-extension method involves amplifying the target sequence with anoutside primer (primer 1) and an internal mutagenesis primer (primer 3),and separately with a second outside primer (primer 4) and an internalprimer (primer 2), yielding two PCR segments (segments A and B). Theinternal mutagenesis primer (primer 3) is designed to contain mismatchesto the target sequence specifying the desired mutation(s). In the secondround of PCR, the products of the first round of PCR (segments A and B)are amplified by PCR using the two outside primers (primers 1 and 4).The resulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector. As the first step of mutagenesis, thestarting DNA (e.g., encoding an Fc fusion protein, an antibody or simplyan Fc region), is operably cloned into a mutagenesis vector. The primersare designed to reflect the desired amino acid substitution. Othermethods useful for the generation of variant Fc regions are known in theart (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351; WO 06/23403).

In some embodiments of the invention, the glycosylation patterns of thebinding members provided herein are modified to enhance ADCC and CDCeffector function. (See Shields R L et al., (JBC. 277:26733-26740, 2002;Shinkawa T et al., JBC. 278:3466-3473, 2003; and Okazaki A et al., J.Mol. Biol., 336:1239, 2004). In some embodiments, an Fc variant proteincomprises one or more engineered glycoforms, i.e., a carbohydratecomposition that is covalently attached to the molecule comprising an Fcregion. Engineered glycoforms may be useful for a variety of purposes,including but not limited to enhancing or reducing effector function.Engineered glycoforms may be generated by any method known to oneskilled in the art, for example by using engineered or variantexpression strains, by co-expression with one or more enzymes, forexample DI N-acetylglucosaminyltransferase III (GnTI11), by expressing amolecule comprising an Fc region in various organisms or cell lines fromvarious organisms, or by modifying carbohydrate(s) after the moleculecomprising Fc region has been expressed. Methods for generatingengineered glycoforms are known in the art, and include but are notlimited to those described in Umana et al, Nat. Biotechnol 17:176-180,1999; Davies et al., Biotechnol Bioeng 74:288-294, 2007; Shields et al,J Biol Chem 277:26733-26740, 2002; Shinkawa et al., J Biol Chem278:3466-3473, 2003) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc.Princeton, N.J.); GlycoMAb™ glycosylation engineering technology(Glycart Biotechnology AG, Zurich, Switzerland). See, e.g., WO00/061739; EA01229125; US 20030115614; Okazaki et al., JMB. 336:1239-49,2004.

Binding members of the invention may be labelled with a detectable orfunctional label. A label can be any molecule that produces or can beinduced to produce a signal, including but not limited to fluorescers,radiolabels, enzymes, chemiluminescers or photosensitizers. Thus,binding may be detected and/or measured by detecting fluorescence orluminescence, radioactivity, enzyme activity or light absorbance.

Suitable labels include, by way of illustration and not limitation,enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase(“G6PDH”) and horseradish peroxidase; dyes; fluorescers, such asfluorescein, rhodamine compounds, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, fluorescamine, fluorophores such aslanthanide cryptates and chelates (Perkin Elmer and C isBiointernational); chemiluminescers such as isoluminol; sensitizers;coenzymes; enzyme substrates; radiolabels including but not limited to¹²⁵I, ¹³¹I, ³⁵S, ³²P, ¹⁴C, ³H, ⁵⁷Co, ⁹⁹TC and ⁷⁵Se and other radiolabelsmentioned herein; particles such as latex or carbon particles; metalsol; crystallite; liposomes; cells, etc., which may be further labelledwith a dye, catalyst or other detectable group. Suitable enzymes andcoenzymes are disclosed in U.S. Pat. No. 4,275,149 and U.S. Pat. No.4,318,980, each of which are herein incorporated by reference in theirentireties. Suitable fluorescers and chemiluminescers are also disclosedin U.S. Pat. No. 4,275,149, which is incorporated herein by reference inits entirety. Labels further include chemical moieties such as biotinthat may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin or streptavidin. Detectable labels may beattached to antibodies of the invention using conventional chemistryknown in the art.

There are numerous methods by which the label can produce a signaldetectable by external means, for example, by visual examination,electromagnetic radiation, heat, and chemical reagents. The label canalso be bound to another binding member that binds the antibody of theinvention, or to a support.

The label can directly produce a signal, and therefore, additionalcomponents are not required to produce a signal. Numerous organicmolecules, for example fluorescers, are able to absorb ultraviolet andvisible light, where the light absorption transfers energy to thesemolecules and elevates them to an excited energy state. This absorbedenergy is then dissipated by emission of light at a second wavelength.This second wavelength emission may also transfer energy to a labelledacceptor molecule, and the resultant energy dissipated from the acceptormolecule by emission of light for example fluorescence resonance energytransfer (FRET). Other labels that directly produce a signal includeradioactive isotopes and dyes.

Alternately, the label may need other components to produce a signal,and the signal producing system would then include all the componentsrequired to produce a measurable signal, which may include substrates,coenzymes, enhancers, additional enzymes, substances that react withenzymic products, catalysts, activators, cofactors, inhibitors,scavengers, metal ions, and a specific binding substance required forbinding of signal generating substances. A detailed discussion ofsuitable signal producing systems can be found in U.S. Pat. No.5,185,243, which is herein incorporated herein by reference in itsentirety.

The binding member, antibody, or one of its functional fragments, can bepresent in the form of an immunoconjugate so as to obtain a detectableand/or quantifiable signal. The immunoconjugates can be conjugated, forexample, with enzymes such as peroxidase, alkaline phosphatase,alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonicanhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase orglucose 6-phosphate dehydrogenase or by a molecule such as biotin,digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can be likewiseconjugated to the immunoconjugates or to their functional fragmentsaccording to the invention and especially include fluorescein and itsderivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone, Lanthanide chelatesor cryptates eg. Europium etc.

The immunoconjugates or their functional fragments can be prepared bymethods known to the person skilled in the art. They can be coupled tothe enzymes or to the fluorescent labels directly or by the intermediaryof a spacer group or of a linking group such as a polyaldehyde, likeglutaraldehyde, ethylenediaminetetraacetic acid (EDTA),diethylene-triaminepentaacetic acid (DPTA), or in the presence ofcoupling agents such as those mentioned above for the therapeuticconjugates. The conjugates containing labels of fluorescein type can beprepared by reaction with an isothiocyanate. Other immunoconjugates canlikewise include chemoluminescent labels such as luminol and thedioxetanes, bio-luminescent labels such as luciferase and luciferin, orelse radioactive labels such as iodine123, iodine-125, iodine-126,iodine-131, iodine-133, bromine-77, technetium99m, indium111, indium113m, gallium67, gallium 68, sulphur35, phosphorus32, carbonl4, tritium(hydrogen3), cobalt57, selenium75, ruthenium95, ruthenium97,ruthenium103, ruthenium105, mercuryl07, mercury203, rhenium99m, rhenium101, rhenium105, scandium47, tellurium121 m, telluriuml22m,telluriuml25m, thuliuml65, thulium167, thuliuml68, fluorine-8, yttrium199. The methods known to the person skilled in the art existing forcoupling the therapeutic radioisotopes to the antibodies either directlyor via a chelating agent such as EDTA, DTPA mentioned above can be usedfor the radioelements which can be used in diagnosis. It is likewisepossible to mention labelling with Na[1125] by the chloraminc T method(Hunter and Greenwood, Nature, 194:495, 1962) or else with technetium99mby the technique of Crockford et al., (U.S. Pat. No. 4,424,200, hereinincorporated by reference in its entirety) or attached via DTPA asdescribed by Hnatowich (U.S. Pat. No. 4,479,930, herein incorporated byreference in its entirety). Further immunoconjugates can include a toxinmoiety such as for example a toxin moiety selected from a group ofPseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof),Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinumtoxin A through F, ricin or a cytotoxic fragment thereof, abrin or acytotoxic fragment thereof, saporin or a cytotoxic fragment thereof,pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1or a cytotoxic fragment thereof.

The present invention provides a method comprising causing or allowingbinding of a binding member as provided herein to IL-4Rα. As noted, suchbinding may take place in vivo, e.g. following administration of abinding member, or nucleic acid encoding a binding member, or it maytake place in vitro, for example in ELISA, Western blotting,immunocytochemistry, immuno-precipitation, affinity chromatography, andbiochemical or cell based assays such as are described herein. Theinvention also provides for measuring levels of antigen directly, byemploying a binding member according to the invention for example in abiosensor system.

For instance, the present invention comprises a method of detectingand/or measuring binding to IL-4Rα, comprising, (i) exposing saidbinding member to IL-4Rα and (ii) detecting binding of said bindingmember to IL-4Rα, wherein binding is detected using any method ordetectable label described herein. This, and any other binding detectionmethod described herein, may be interpreted directly by the personperforming the method, for instance, by visually observing a detectablelabel. Alternatively, this method, or any other binding detection methoddescribed herein, may produce a report in the form of an autoradiograph,a photograph, a computer printout, a flow cytometry report, a graph, achart, a test tube or container or well containing the result, or anyother visual or physical representation of a result of the method.

The amount of binding of binding member to IL-4Rα may be determined.Quantification may be related to the amount of the antigen in a testsample, which may be of diagnostic interest. Screening for IL-4Rαbinding and/or the quantification thereof may be useful, for instance,in screening patients for diseases or disorders associated with IL-4Rα,such as are referred to elsewhere herein. In one embodiment, amongothers, a diagnostic method of the invention comprises (i) obtaining atissue or fluid sample from a subject, (ii) exposing said tissue orfluid sample to one or more binding members of the present invention;and (iii) detecting bound IL-4Rα as compared to a control sample,wherein an increase in the amount of IL-4Rα binding as compared to thecontrol may indicate an aberrant level of IL-4Rα expression or activity.Tissue or fluid samples to be tested include blood, serum, urine, biopsymaterial, tumours, or any tissue suspected of containing aberrant IL-4Rαlevels. Subjects testing positive for aberrant IL-4Rα levels or activitymay also benefit from the treatment methods disclosed later herein.

Those skilled in the art are able to choose a suitable mode ofdetermining binding of the binding member to an antigen according totheir preference and general knowledge, in light of the methodsdisclosed herein.

The reactivities of binding members in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the binding member. Bound antigen isphysically separated from unbound antigen and the amount of radioactiveantigen bound to the binding member determined. The more antigen thereis in the test sample the less radioactive antigen will bind to thebinding member. A competitive binding assay may also be used withnon-radioactive antigen, using antigen or an analogue linked to areporter molecule. The reporter molecule may be a fluorochrome, phosphoror laser dye with spectrally isolated absorption or emissioncharacteristics. Suitable fluorochromes include fluorescein, rhodamine,phycoerythrin, Texas Red, and lanthanide chelates or cryptates. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes, which catalyze reactions that develop, or change colours orcause changes in electrical properties, for example. They may bemolecularly excitable, such that electronic transitions between energystates result in characteristic spectral absorptions or emissions. Theymay include chemical entities used in conjunction with biosensors.Biotin/avidin or biotin/streptavidin and alkaline phosphatase detectionsystems may be employed.

The signals generated by individual binding member-reporter conjugatesmay be used to derive quantifiable absolute or relative data of therelevant binding member binding in samples (normal and test).

A kit comprising a binding member according to any aspect or embodimentof the present invention is also provided as an aspect of the presentinvention. In the kit, the binding member may be labelled to allow itsreactivity in a sample to be determined, e.g. as described furtherbelow. Further the binding member may or may not be attached to a solidsupport. Components of a kit are generally sterile and in sealed vialsor other containers. Kits may be employed in diagnostic analysis orother methods for which binding members are useful. A kit may containinstructions for use of the components in a method, e.g. a method inaccordance with the present invention. Ancillary materials to assist inor to enable performing such a method may be included within a kit ofthe invention. The ancillary materials include a second, differentbinding member, which binds to the first binding member and isconjugated to a detectable label (e.g., a fluorescent label, radioactiveisotope or enzyme). Antibody-based kits may also comprise beads forconducting an immunoprecipitation. Each component of the kits isgenerally in its own suitable container. Thus, these kits generallycomprise distinct containers suitable for each binding member. Further,the kits may comprise instructions for performing the assay and methodsfor interpreting and analyzing the data resulting from the performanceof the assay.

The present invention also provides the use of a binding member as abovefor measuring antigen levels in a competition assay, that is to say amethod of measuring the level of antigen in a sample by employing abinding member as provided by the present invention in a competitionassay. This may be where the physical separation of bound from unboundantigen is not required. Linking a reporter molecule to the bindingmember so that a physical or optical change occurs on binding is onepossibility. The reporter molecule may directly or indirectly generatedetectable signals, which may be quantifiable. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

For example, the present invention includes a method of identifying anIL-4Rα binding compound, comprising (i) immobilizing IL-4Rα to asupport, (ii) contacting said immobilized IL-4Rα simultaneously or in astep-wise manner with at least one tagged or labelled binding memberaccording to the invention and one or more untagged or unlabelled testbinding compounds, and (iii) identifying a new IL-4Rα binding compoundby observing a decrease in the amount of bound tag from the taggedbinding member.

An alternative method of identifying an IL-4Rα binding compound maycomprise (i) immobilizing binding member to a support, (ii) contactingsaid immobilized binding member simultaneously or in a step-wise mannerwith tagged IL-4Rα and one or more untagged or unlabelled test bindingmembers or binding compounds, (iii) identifying a new IL-4Rα bindingcompound by observing a decrease in the amount of bound tag from thetagged IL-4Rα.

Such methods can be performed in a high-throughput manner using amultiwell or array format. Such assays may also be performed in solutionfor example as an HTRF® assay as described in Example 4.3. See, forinstance, U.S. Pat. No. 5,814,468, which is herein incorporated byreference in its entirety. As described above, detection of binding maybe interpreted directly by the person performing the method, forinstance, by visually observing a detectable label, or a decrease in thepresence thereof. Alternatively, the binding methods of the inventionmay produce a report in the form of an autoradiograph, a photograph, acomputer printout, a flow cytometry report, a graph, a chart, a testtube or container or well containing the result, or any other visual orphysical representation of a result of the method.

Competition assays can also be used in epitope mapping. In one instanceepitope mapping may be used to identify the epitope bound by an IL-4Rαbinding member, which optionally may have optimized neutralizing and/ormodulating characteristics. Such an epitope can be linear orconformational. A conformational epitope can comprise at least twodifferent fragments of IL-4Rα, wherein said fragments are positioned inproximity to each other when IL-4Rα is folded in its tertiary orquaternary structure to form a conformational epitope which isrecognized by an inhibitor of IL-4Rα, such as an IL-4Rα binding member.In testing for competition a peptide fragment of the antigen may beemployed, especially a peptide including or consisting essentially of anepitope of interest. A peptide having the epitope sequence plus one ormore amino acids at either end may be used. Binding members according tothe present invention may be such that their binding for antigen isinhibited by a peptide with or including the sequence given.

The present invention further provides an isolated nucleic acid encodinga binding member of the present invention. Nucleic acid may include DNAand/or RNA. In one aspect, the present invention provides a nucleic acidthat codes for a CDR or set of CDRs or VH domain or VL domain orantibody antigen-binding site or antibody molecule, e.g. scFv or IgG,e.g. IgG1, IgG2 or IgG4, of the invention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell thatcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDRs or VH domain or VL domain or antibody antigen-bindingsite or antibody molecule, e.g. scFv or IgG1, IgG2 or IgG4 as provided,itself forms an aspect of the present invention, as does a method ofproduction of the encoded product, which method comprises expressionfrom encoding nucleic acid. Expression may conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression a VH or VL domain,or binding member may be isolated and/or purified using any suitabletechnique, then used as appropriate.

Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and bindingmembers comprising a VH and/or VL domain are provided as further aspectsof the present invention.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals. The expression ofantibodies and antibody fragments in prokaryotic cells is wellestablished in the art. For a review, see for example Pliickthun 1991. Acommon bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a binding member forexample Chadd & Chamow (Current Opinion in Biotechnology 12:188-194,2001), Andersen & Krummen (Current Opinion in Biotechnology 13:117,2002) and Larrick & Thomas (Current Opinion in Biotechnology 12:411-418,2001). Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 ratmyeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage, as appropriate. For further details see,for example, Sambrook & Russell (Molecular Cloning a Laboratory Manual:3rd edition, 2001, Cold Spring Harbor Laboratory Press). Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Ausubel et al. eds., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, John Wiley & Sons, 4^(th) edition 1999.

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intracellular expression of thebinding members of the present invention as “intrabodies” orintracellular antibodies. Intrabodies may be used for gene therapy.

A still further aspect provides a method comprising introducing nucleicacid of the invention into a host cell. The introduction may employ anyavailable technique. For eukaryotic cells, suitable techniques mayinclude calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus.Introducing nucleic acid in the host cell, in particular a cukaryoticcell may use a viral or a plasmid based system. The plasmid system maybe maintained episomally or may incorporated into the host cell or intoan artificial chromosome. Incorporation may be either by random ortargeted integration of one or more copies at single or multiple loci.For bacterial cells, suitable techniques may include calcium chloridetransformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. The purification of the expressed product may beachieved by methods known to one of skill in the art.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express abinding member or polypeptide as above.

Binding members of the present invention may be used in methods ofdiagnosis or treatment in human or animal subjects, e.g. human. Forinstance, binding members may be used in diagnosis or treatment ofIL-4Rα-associated diseases or disorders, examples of which are referredto elsewhere herein.

Particular conditions for which a binding member of the invention may beused in treatment or diagnosis include: asthma, COPD (including chronicbronchitis, small airway disease and emphysema), inflammatory boweldisease, fibrotic conditions (including systemic sclerosis, pulmonaryfibrosis, parasite-induced liver fibrosis, and cystic fibrosis, allergy(including for example atopic dermatitis and food allergy), transplationtherapy to prevent transplant rejection, as well as suppression ofdelayed-type hypersensitivity or contact hypersensitivity reactions, asadjuvants to allergy immunotherapy and as vaccine adjuvants.

Thus, binding members of the invention are useful as therapeutic agentsin the treatment of conditions involving IL-4, IL-13 or IL-4Rαexpression and/or activity. One embodiment, among others, is a method oftreatment comprising administering an effective amount of a bindingmember of the invention to a patient in need thereof, wherein functionalconsequences of IL-4Rα activation are decreased. Another embodiment,among others, is a method of treatment comprising (i) identifying apatient demonstrating IL-4, IL-13 or IL-4Rα expression or activity, forinstance using the diagnostic methods described above, and (ii)administering an effective amount of a binding member of the inventionto the patient, wherein the functional consequences of IL-4Rα activationare attenuated. An effective amount according to the invention is anamount that modulates (e.g. decreases) the functional consequences ofIL-4Rα activation so as to modulate (e.g. decrease or lessen) theseverity of at least one symptom of the particular disease or disorderbeing treated, but not necessarily cure the disease or disorder.Accordingly, one embodiment of the invention is a method of treating orreducing the severity of at least one symptom of any of the disordersreferred to herein, comprising administering to a patient in needthereof an effective amount of one or more binding members of thepresent invention alone or in a combined therapeutic regimen withanother appropriate medicament known in the art or described herein suchthat the severity of at least one symptom of any of the disorders isreduced. Another embodiment of the invention, among others, is a methodof antagonizing at least one effect of IL-4Rα comprising contacting withor administering an effective amount of one or more binding members ofthe present invention such that said at least one effect of IL-4Rα isantagonized, e.g. the ability of IL-4Rα to form a complex (the precursorto active signalling) with IL-4.

Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a binding member as provided, orpharmaceutical compositions comprising such a binding member, and/or useof such a binding member in the manufacture of a medicament foradministration, for example in a method of making a medicament orpharmaceutical composition comprising formulating the binding memberwith a pharmaceutically acceptable excipient. A pharmaceuticallyacceptable excipient may be a compound or a combination of compoundsentering into a pharmaceutical composition not provoking secondaryreactions and which allows, for example, facilitation of theadministration of the active compound(s), an increase in its lifespanand/or in its efficacy in the body, an increase in its solubility insolution or else an improvement in its conservation. Thesepharmaceutically acceptable vehicles are well known and will be adaptedby the person skilled in the art as a function of the nature and of themode of administration of the active compound(s) chosen.

Binding members of the present invention will usually be administered inthe form of a pharmaceutical composition, which may comprise at leastone component in addition to the binding member. Thus pharmaceuticalcompositions according to the present invention, and for use inaccordance with the present invention, may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, inhaled or by injection, e.g. intravenous. In on embodiment thecomposition is sterile.

Pharmaceutical compositions for oral administration such as for examplenanobodies etc are also envisaged in the present invention. Such oralformulations may be in tablet, capsule, powder, liquid or semi-solidform. A tablet may comprise a solid carrier such as gelatin or anadjuvant. Liquid pharmaceutical compositions generally comprise a liquidcarrier such as water, petroleum, animal or vegetable oils, mineral oilor synthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be employed, as required, including buffers such asphosphate, citrate, histidine and other organic acids; antioxidants suchas ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agentssuch as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

Binding members of the present invention may be formulated in liquid,semi-solid or solid forms depending on the physicochemical properties ofthe molecule and the route of delivery. Formulations may includeexcipients, or combinations of excipients, for example: sugars, aminoacids and surfactants. Liquid formulations may include a wide range ofantibody concentrations and pH. Solid formulations may be produced bylyophilisation, spray drying, or drying by supercritical fluidtechnology, for example. Formulations of anti-IL-4Rα will depend uponthe intended route of delivery: for example, formulations for pulmonarydelivery may consist of particles with physical properties that ensurepenetration into the deep lung upon inhalation; topical formulations mayinclude viscosity modifying agents, which prolong the time that the drugis resident at the site of action. In certain embodiments, the bindingmember may be prepared with a carrier that will protect the bindingmember against rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are known to those skilled in the art. See, e.g.,Robinson, 1978.

Anti-IL-4Rα treatment with a binding member of the invention may begiven orally (for example nanobodies) by injection (for example,subcutaneously, intra-articular, intravenously, intraperitoneal,intra-arterial or intramuscularly), by inhalation, by the intravesicularroute (instillation into the urinary bladder), or topically (for exampleintraocular, intranasal, rectal, into wounds, on skin). The treatmentmay be administered by pulse infusion, particularly with declining dosesof the binding member. The route of administration can be determined bythe physicochemical characteristics of the treatment, by specialconsiderations for the disease or by the requirement to optimizeefficacy or to minimize side-effects. One particular route ofadministration is intravenous. Another route of administeringpharmaceutical compositions of the present invention is subcutaneously.It is envisaged that anti-IL-4Rα treatment will not be restricted to usein hospitals or doctor's offices but rather may include homes and placesof work. Therefore, subcutaneous injection using a needle-free device isadvantageous.

A composition may be administered alone or in combination with othertreatments, concurrently or sequentially or as a combined preparationwith another therapeutic agent or agents, dependent upon the conditionto be treated.

A binding member for IL-4Rα may be used as part of a combination therapyin conjunction with an additional medicinal component. Combinationtreatments may be used to provide significant synergistic effects,particularly the combination of an anti-IL-4Rα binding member with oneor more other drugs. A binding member for IL-4Rα may be administeredconcurrently or sequentially or as a combined preparation with anothertherapeutic agent or agents, for the treatment of one or more of theconditions listed herein.

A binding member according to the present invention may be provided incombination or addition with one or more of the following agents:

a cytokine or agonist or antagonist of cytokine function (e.g. an agentwhich acts on cytokine signalling pathways, such as a modulator of theSOCS system), such as an alpha-, beta- and/or gamma-interferon;insulin-like growth factor type I (IGF-1), its receptors and associatedbinding proteins; interleukins (IL), e.g. one or more of IL-1 to -33,and/or an interleukin antagonist or inhibitor, such as anakinra;inhibitors of receptors of interleukin family members or inhibitors ofspecific subunits of such receptors, a tumour necrosis factor alpha(TNF-α) inhibitor, such as an anti-TNF monoclonal antibodies (forexample infliximab, adalimumab and/or CDP-870) and/or a TNF receptorantagonist, e.g. an immunoglobulin molecule (such as etanercept) and/ora low-molecular-weight agent, such as pentoxyfylline;

a modulator of B cells, e.g. a monoclonal antibody targetingB-lymphocytes (such as CD20 (rituximab) or MRA-aIL16R) or T-lymphocytes(e.g. CTLA4-Tg or Abatacept);

a modulator that inhibits osteoclast activity, for example an antibodyto RANKL;

-   -   a modulator of chemokine or chemokine receptor function, such as        an antagonist of CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5,        CCR6, CCR7, CCR8, CCR9, CCR10 or CCR11 (for the C-C family);        CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCL13 (for the        C-X-C family) or CX₃CR1 (for the C-X₃-C family);

an inhibitor of matrix metalloproteases (MMPs), i.e. one or more of thestromelysins, the collagenases and the gelatinases as well asaggrecanasc, especially collagenase-1 (MMP-1), collagenase-2 (MMP-8),collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10)and/or stromelysin-3 (MMP-11) and/or MMP-9 and/or MMP-12, e.g. an agentsuch as doxycycline;

a leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO) inhibitor or5-lipoxygenase activating protein (FLAP) antagonist, such as zileuton;ABT-761; fenleuton; tepoxalin; Abbott-79175; Abbott-85761;N-(5-substituted)-thiophene-2-alkylsulfonamides;2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such asZeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted2-cyanonaphthalene compound, such as L-739,010; a 2-cyanoquinolinccompound, such as L-746,530; indolc and/or a quinolinc compound, such asMK-591, MK-886 and/or BAY x 1005;

a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4,selected from the group consisting of the phenothiazin-3-1s, such asL-651,392; amidino compounds, such as CGS-25019c; benzoxalamines, suchas ontazolast; benzenecarboximidamides, such as BIIL 284/260; andcompounds, such as zafirlukast, ablukast, montelukast, pranlukast,verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A) and BAYx 7195;

a phosphodiesterase (PDE) inhibitor, such as a methylxanthanine, e.g.theophylline and/or aminophylline; and/or a selective PDE isoenzymeinhibitor, e.g. a PDE4 inhibitor and/or inhibitor of the isoform PDE4Dand/or an inhibitor of PDE5;

-   -   a histamine type 1 receptor antagonist, such as cetirizine,        loratadine, desloratadine, fexofenadine, acrivastine,        terfenadine, astemizole, azelastine, levocabastine,        chlorpheniramine, promethazine, cyclizine, and/or mizolastine        (generally applied orally, topically or parenterally);

a proton pump inhibitor (such as omeprazole) or gastroprotectivehistamine type 2 receptor antagonist;

an antagonist of the histamine type 4 receptor;

an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimeticagent, such as propylhexedrine, phenylephrine, phenylpropanolamine,ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazolinehydrochloride, tetrahydrozoline hydrochloride, xylometazolinehydrochloride, tramazoline hydrochloride and ethylnorepinephrinehydrochloride;

an anticholinergic agent, e.g. a muscarinic receptor (e.g. M1, M2, M3,M4 or M5) antagonist, such as atropine, hyoscine, glycopyrrrolate,ipratropium bromide, tiotropium bromide, oxitropium bromide, pirenzepineand telenzepine;

a beta-adrenoceptor agonist (including beta receptor subtypes 1-4), suchas isoprenaline, salbutamol, formoterol, salmeterol, terbutaline,orciprenaline, bitolterol mesylate and/or pirbuterol, e.g. a chiralenantiomer thereof;

a chromone, e.g. sodium cromoglycate and/or nedocromil sodium;

a glucocorticoid, such as flunisolide, triamcinolone acetonide,beclomethasone dipropionatc, budesonidc, fluticasonc propionate,cicicsonidc, and/or momctasonc furoatc;

an agent that modulate nuclear hormone receptors, such as a PPAR;

an immunoglobulin (Ig) or Ig preparation or an antagonist or antibodymodulating Ig function, such as anti-IgE (e.g. omalizumab);

other systemic or topically-applied anti-inflammatory agent, e.g.thalidomide or a derivative thereof; a retinoid, dithranol and/orcalcipotriol;

combinations of aminosalicylates and sulfapyridine, such assulfasalazine, mesalazine, balsalazide, and olsalazine; andimmunomodulatory agents, such as the thiopurines; and corticosteroids,such as budesonide;

an antibacterial agent, e.g. a penicillin derivative, a tetracycline, amacrolide, a beta-lactam, a fluoroquinolone, metronidazole and/or aninhaled aminoglycoside; and/or an antiviral agent, e.g. acyclovir,famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine,rimantadine; ribavirin; zanamavir and/or oseltamavir; a proteaseinhibitor, such as indinavir, nelfinavir, ritonavir and/or saquinavir; anucleoside reverse transcriptase inhibitor, such as didanosine,lamivudine, stavudine, zalcitabine, zidovudine; a non-nucleoside reversetranscriptase inhibitor, such as nevirapine, efavirenz;

a cardiovascular agent, such as a calcium channel blocker,beta-adrenoceptor blocker, angiotensin-converting enzyme (ACE)inhibitor, angiotensin-2 receptor antagonist; lipid lowering agent, suchas a statin and/or fibrate; a modulator of blood cell morphology, suchas pentoxyfylline; a thrombolytic and/or an anticoagulant, e.g. aplatelet aggregation inhibitor;

a CNS agent, such as an antidepressant (such as sertraline),anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole,pramipexole; MAOB inhibitor, such as selegine and rasagiline; comPinhibitor, such as tasmar; A-2 inhibitor, dopamine reuptake inhibitor,NMDA antagonist, nicotine agonist, dopamine agonist and/or inhibitor ofneuronal nitric oxide synthase) and an anti-Alzheimer's drug, such asdonepezil, rivastigmine, tacrine, COX-2 inhibitor, propentofylline ormetrifonate;

an agent for the treatment of acute and chronic pain, e.g. a centrallyor peripherally-acting analgesic, such as an opioid analogue orderivative, carbamazepine, phenyloin, sodium valproate, amitryptiline orother antidepressant agent, paracetamol, or non-steroidalanti-inflammatory agent;

a parenterally or topically-applied (including inhaled) localanaesthetic agent, such as lignocaine or an analogue thereof;

an anti-osteoporosis agent, e.g. a hormonal agent, such as raloxifene,or a biphosphonate, such as alendronate;

(i) a tryptase inhibitor; (ii) a platelet activating factor (PAF)antagonist; (iii) an interleukin converting enzyme (ICE) inhibitor; (iv)an IMPDH inhibitor; (v) an adhesion molecule inhibitors including VLA-4antagonist; (vi) a cathepsin; (vii) a kinase inhibitor, e.g. aninhibitor of tyrosine kinases (such as Btk, Itk, Jak3 MAP examples ofinhibitors might include Gefitinib, Imatinib mesylate), aserine/threonine kinase (e.g. an inhibitor of MAP kinase, such as p38,JNK, protein kinases A, B and C and IKK), or a kinase involved in cellcycle regulation (e.g. a cylin dependent kinase); (viii) a glucose-6phosphate dehydrogenase inhibitor; (ix) a kinin-B.sub1.- and/orB.sub2.-receptor antagonist; (x) an anti-gout agent, e.g. colchicine;(xi) a xanthine oxidase inhibitor, e.g. allopurinol; (xii) a uricosuricagent, e.g. probenecid, sulfinpyrazone, and/or benzbromarone; (xiii) agrowth hormone secretagogue; (xiv) transforming growth factor (TGFI3);(xv) platelet-derived growth factor (PDGF); (xvi) fibroblast growthfactor, e.g. basic fibroblast growth factor (bFGF); (xvii) granulocytemacrophage colony stimulating factor (GM-CSF); (xviii) capsaicin cream;(xix) a tachykinin NK.sub1. and/or NK.sub3. receptor antagonist, such asNKP-608C, SB-233412 (talnetant) and/or D-4418; (xx) an elastaseinhibitor, e.g. UT-77 and/or ZD-0892; (xxi) a TNF-alpha convertingenzyme inhibitor (TACE); (xxii) induced nitric oxide synthase (iNOS)inhibitor or (xxiii) a chemoattractant receptor-homologous moleculeexpressed on TH2 cells (such as a CRTH2 antagonist); (xxiv) an inhibitorof a P38 (xxv) agent modulating the function of Toll-like receptors(TLR) and (xxvi) an agent modulating the activity of purinergicreceptors, such as P2×7; (xxvii) an inhibitor of transcription factoractivation, such as NFkB, API, and/or STATS.

An inhibitor may be specific or may be a mixed inhibitor, e.g. aninhibitor targeting more than one of the molecules (e.g. receptors) ormolecular classes mentioned above.

The binding member could also be used in association with achemotherapeutic agent or another tyrosine kinase inhibitor inco-administration or in the form of an immunoconjugate. Fragments ofsaid antibody could also be use in bispecific antibodies obtained byrecombinant mechanisms or biochemical coupling and then associating thespecificity of the above described antibody with the specificity ofother antibodies able to recognize other molecules involved in theactivity for which IL-4Rα is associated.

For treatment of an inflammatory disease, e.g. rheumatoid arthritis,osteoarthritis, asthma, allergic rhinitis, chronic obstructive pulmonarydisease (COPD), or psoriasis, a binding member of the invention may becombined with one or more agents, such as non-steroidalanti-inflammatory agents (hereinafter NSAIDs) including non-selectivecyclo-oxygenase (COX)-1/COX-2 inhibitors whether applied topically orsystemically, such as piroxicam, diclofenac, propionic acids, such asnaproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates,such as mefenamic acid, indomethacin, sulindac, azapropazone,pyrazolones, such as phenylbutazone, salicylates, such as aspirin);selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib,valdecoxib, lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenaseinhibiting nitric oxide donors (CINODs); glucocorticosteroids (whetheradministered by topical, oral, intra-muscular, intra-venous orintra-articular routes); methotrexate, leflunomide; hydroxychloroquine,d-penicillamine, auranofin or other parenteral or oral goldpreparations; analgesics; diacerein; intra-articular therapies, such ashyaluronic acid derivatives; and nutritional supplements, such asglucosamine.

A binding member of the invention can also be used in combination withan existing therapeutic agent for the treatment of cancer. Suitableagents to be used in combination include:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as Gleevec (imatinib mesylate),alkylating agents (for example cis-platin, carboplatin,cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphanand nitrosoureas); antimetabolites (for example antifolates, such asfluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, hydroxyurea, gemcitabine andpaclitaxel); antitumour antibiotics (for example anthracyclines likeadriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin,mitomycin-C, dactinomycin and mithramycin); antimitotic agents (forexample vinca alkaloids like vincristinc, vinblastinc, vindesine andvinorelbine and taxoids like taxol and taxotere); and topoisomeraseinhibitors (for example epipodophyllotoxins like etoposide andteniposide, amsacrine, topotecan and camptothecins);(ii) cytostatic agents, such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goscrelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase, such as finasteride;(iii) Agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-erbb2 antibody trastuzumab and the anti-erbb1 antibodycetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinaseinhibitors and serine/threonine kinase inhibitors, for exampleinhibitors of the epidermal growth factor family (for example EGFRfamily tyrosine kinase inhibitors, such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033)), for example inhibitors of the platelet-derived growth factorfamily and for example inhibitors of the hepatocyte growth factorfamily;(v) antiangiogenic agents, such as those which inhibit the effects ofvascular endothelial growth factor (for example the anti-vascularendothelial cell growth factor antibody bevacizumab, compounds, such asthose disclosed in International Patent Applications WO 97/22596, WO97/30035, WO 97/32856 and WO 98/13354, each of which is incorporatedherein in its entirety) and compounds that work by other mechanisms (forexample linomide, inhibitors of integrin avf33 function andangiostatin);(vi) vascular damaging agents, such as combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213 (each of which isincorporated herein in its entirety);(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;(viii) gene therapy approaches, including for example approaches toreplace aberrant genes, such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene directed enzyme pro-drug therapy) approaches, such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy, such as multi-drug resistance gene therapy; and(ix) immunotherapeutic approaches, including for example ex vivo and invivo approaches to increase the immunogenicity of patient tumour cells,such as transfection with cytokines, such as interleukin 2, interleukin4 or granulocyte macrophage colony stimulating factor, approaches todecrease T-cell anergy, approaches using transfected immune cells, suchas cytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies.

A binding member of the invention and one or more of the aboveadditional medicinal components may be used in the manufacture of amedicament. The medicament may be for separate or combinedadministration to an individual, and accordingly may comprise thebinding member and the additional component as a combined preparation oras separate preparations. Separate preparations may be used tofacilitate separate and sequential or simultaneous administration, andallow administration of the components by different routes e.g. oral andparenteral administration.

In accordance with the present invention, compositions provided may beadministered to mammals. Administration may be in a “therapeuticallyeffective amount”, this being sufficient to show benefit to a patient.Such benefit may be at least amelioration of at least one symptom. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe composition, the type of binding member, the method ofadministration, the scheduling of administration and other factors knownto medical practitioners. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and may depend on the severity of the symptomsand/or progression of a disease being treated. Appropriate doses ofantibody are well known in the art (Ledermann et al. Int. J. Cancer47:659-664, 1991; Bagshawc et al. Antibody, Immunoconjugates andRadiopharmaceuticals 4:915-922, 1991). Specific dosages indicatedherein, or in the Physician's Desk Reference (2003) as appropriate forthe type of medicament being administered, may be used. Atherapeutically effective amount or suitable dose of a binding member ofthe invention can be determined by comparing its in vitro activity andin vivo activity in an animal model. Methods for extrapolation ofeffective dosages in mice and other test animals to humans arc known.The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis, prevention or for treatment, the size andlocation of the area to be treated, the precise nature of the antibody(e.g. whole antibody, fragment or diabody), and the nature of anydetectable label or other molecule attached to the antibody. A typicalantibody dose will be in the range 100 μg to 1 g for systemicapplications, and 1 μg to 1 mg for topical applications. An initialhigher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 isotype. This is a dose for a single treatment of an adult patient,which may be proportionally adjusted for children and infants, and alsoadjusted for other antibody formats in proportion to molecular weight.Treatments may be repeated at daily, twice-weekly, weekly or monthlyintervals, at the discretion of the physician. Treatments may be everytwo to four weeks for subcutaneous administration and every four toeight weeks for intravenous administration. In some embodiments of thepresent invention, treatment is periodic, and the period betweenadministrations is about two weeks or more, e.g. about three weeks ormore, about four weeks or more, or about once a month. In otherembodiments of the invention, treatment may be given before, and/orafter surgery, and may be administered or applied directly at theanatomical site of surgical treatment.

The invention will be further described by following non-limitingExamples and

Figures in which:

FIG. 1 shows alignment of the VH domains of Antibodies 2-42 againstAntibody 1 (split into sheets a and b).

FIG. 2 shows alignment of the VL domains of Antibodies 2-42 againstAntibody 1 (split into sheets a and b).

FIG. 3 shows alignment of VH domains of Antibodies 1-19 and 21-42against Antibody 20 (split into sheets a and b).

FIG. 4 shows alignment of VL domains of Antibodies 1-19 and 21-42against Antibody 20 (split into sheets a and b).

FIG. 5 is sequence identity tables for 6xCDRs (split into a, b and c).

FIG. 6 is sequence identity tables for 3xVH CDRs (split into a, b andc).

FIG. 7 is sequence identity tables for 3xVL CDRs (split into a, b andc).

EXAMPLES Example 1 1.1 Cloning of human IL-4Rα Extracellular Domain

A cDNA encoding the sequence of human IL-4Rα extracellular domain (aminoacid residues 1-229 Swiss-Prot Accession number P24394) was amplifiedfrom HUVEC cDNA library via PCR using primers based on the human IL-4RαcDNA sequence (RefSeq NM_(—)000418). The resulting cDNA was sub-clonedfollowing the manufacture's instructions into pDONR201 (Invitrogen).

The cDNA fragments coding the IL-4Rα extracellular domains were thentransferred to mammalian expression vector pDEST12.2 (Invitrogen) usingLR Gateway® reaction (Invitrogen). The pDEST12.2 vector had beenmodified to contain the human IgG1 Fc coding region, polyhistidine(His6) tag in-frame with the inserted gene of interest, and also byinsertion of the oriP origin of replication from the pCEP4 vector(Invitrogen) allowing episomal plasmid replication upon transfectioninto cell lines expressing the EBNA-1 gene product (such as HEK293-EBNAcells).

The public database accession number for human IL-4Rα mRNA is NM-000418;the key region of interest with this database sequence is 243-929. Thepredicted amino acid sequence for the resultant human IL-4Rα/Fc is shownin SEQ ID NO: 454.

1.1.1 Expression and Purification

HEK-EBNA cells were transfected using PEI. Protein was purified fromconditioned media using Protein G chromatography followed by SizeExclusion chromatography.

1.2 Cloning of Recombinant Cynomolgus Monkey IL-4Rα and Human IL-4Rα175V Mutant

The cynomolgus monkey IL-4Rα subunit was amplified from cynomolgousmonkey thymus and lymph node (BioCat GmbH) via the polymerase chainreaction (PCR) using the following oligonucleotides as primers:

(SEQ ID NO: 451) 5′ ggggacaagt ttgtacaaaa aagcaggctt ctttaactttaagaaggaga tataaccatg gggtggcttt gctctgggct cctgttgcct gtgagc-3′(SEQ ID NO: 452) 5′-ggggaccact ttgtacaaga aagctgggtc ctgctcgaagggctccctgt aggagttgta cca-3′

The resulting cDNA was sub-cloned following the manufacturer'sinstructions into pDONR201 (Invitrogen). The sequence of the cynomolgusmonkey IL-4Rα extracellular domain is shown in SEQ ID NO: 455.

1.3 Cloning human IL-4Rα 175V Variant

The polymorphism of the human IL-4Rα, 175V was generated using thepDONR201 vector containing the coding sequence for the human IL-4Rα(amino acid residues 1-229 NP_(—)000409). The isoleucine at amino acidposition 75 was mutated to valine using the QuikChange Multisite-directed mutagenesis kit (Stratagene) using the followingoligonucleotide as mutation primer:5′-gaagcccacacgtgtgccctgagaacaacgga-3′ (SEQ ID NO: 453)

1.4 Generation of Recombinant Baculovirus for Cynomolgus and 175VVariant IL-4Rα/Fc

Cynomolgus monkey IL-4Rα and human IL-4Rα 175V variant, were theninserted into a Gateway adapted pFastBac vector (in-house) containing ahuman IgG1 Fc coding region. Nucleotide and protein sequences forcynomolgus IL4Rα/Fc are shown in SEQ ID NO: 456 and SEQ ID NO: 457,respectively. Nucleotide and protein sequences for 175V IL4Rα/Fc areshown in SEQ ID NO: 458 and SEQ ID NO: 459, respectively. Generation ofrecombinant Bacmid was done by transformation of DH10Bac E. coli(Invitrogen) and plated on LB agar with selection medium. Sf9 cells weretransfected with recombinant bacmids, and high titre recombinantbaculovirus were produced.

1.5 Expression and Purification of Human 175V and Cynomolgus Fc TaggedIL-4Rα Protein Variants

Proteins were expressed in Sf21 cells (400 ml) infected with virus MOI 1at cell density of 3×10⁶ cells/ml in SF-900 II SFM media (Invitrogen).Media containing the secreted IL-4Rα-Fc fusion proteins were harvestedafter 72 and 96 hours, respectively. The growth medium from the Sf21cells (400 ml) was adjusted to pH 8.0. Protein was purified usingProtein G chromatography followed by Size Exclusion chromatography.

Example 2 Lead Isolation 2.1 Selections

Naïve human single chain Fv (scFv) phage display libraries cloned in toa phagemid vector based on the filamentous phage M13 were used forselections (Vaughan et al. Nature Biotechnology 14(3):309-314, 1996;Hutchings, C. Generation of Naïve Human Antibody Libraries, in AntibodyEngineering, R. Kontermann and S. Dubel, Editors. 2001, SpringerLaboratory Manuals, Berlin. p. 93). Anti-IL-4Rα specific scFv antibodieswere isolated from the phage display libraries using a series ofselection cycles on recombinant human IL-4Rα Fc (R & D Systems)essentially as previously described by Vaughan et al (Vaughan, T J. etal. Nature Biotechnology 14(3):309-14, 1996) and Hawkins et al. Journalof Molecular Biology 226:889-896, 1992). In brief, for panningselections, human IL-4Rα Fc in PBS (Dulbecco's PBS, pH7.4) was adsorbedonto wells of an microtitre plate (Nunc) overnight at 4° C. Wells werewashed with PBS then blocked for 1 h with PBS-Marvel (3% w/v). Purifiedphage in PBS-Marvel (3% w/v), containing a 10 fold excess of irrelevantFc tagged protein, were added to the wells and allowed to bind coatedantigen for 1 h. Unbound phage was removed by a series of wash cyclesusing PBS-Tween (0.1% v/v) and PBS. Bound phage particles were eluted,infected into E. coli TG1 bacteria and rescued for the next round ofselection (Vaughan et al. Nature Biotechnology 14(3):309-314, 1996).

2.2 Inhibition of IL4 Binding to IL-4 Receptor by Unpurified scFv

Unpurified scFv from periplasmic preparations were screened in twohomogeneous time-resolved fluorescence (HTRF®) receptor-ligand bindingassays, run in parallel to measure inhibitory activity against bothhuman and cynomolgus IL4Rα. In the human assay, unpurified scFv samplescompeted with human biotinylated IL4 (Peprotec with in-housebiotinylation) for binding to human IL4Rα-Fc receptor (R and D Systems,604-4R). In the cynomolgus assay, unpurified scFv samples competed withcynomolgus biotinylated IL4 (in-house E. coli expressed withbiotinylation in-house) for binding to cynomolgus IL4Rα-Fc-HIS6(in-house HEK expressed). The detailed assay methods are provided in theMaterials and Methods section (2.4).

ScFv which showed an inhibitory effect as unpurified periplasmicextracts, on the binding of IL4 to IL4Rα in both human and cynomolgusreceptor-ligand assays, were subjected to DNA sequencing (see: Osbournet al. Immunotechnology. 2:181-196, 1996). ScFvs with unique sequenceswere expressed in bacteria and purified by affinity chromatography (asdescribed by Bannister et al. Biotechnology and bioengineering, 94.931-937, 2006).

2.3 Inhibition of IL-4 Binding to IL-4 Receptor by Purified scFv

Potency of scFv samples was determined by competing a dilution series ofthe purified scFv preparation against IL4 for binding to IL4Rα. Bothhuman and cynomolgus receptor-ligand assays were performed in parallelas described in the Materials and Methods section 2.4. Purified scFvpreparations of Antibody 1 inhibited binding of human IL4 to the IL4Rαwith a K, value of 12 nM (95% C18.7, 16.6) Inhibition of cynomolgus IL4binding to IL4Rα by Antibody 1 was incomplete with 10% inhibition ofassay signal observed. It was therefore not possible to calculateaccurate K, potency data from the results obtained.

2.4 Materials and methods

Receptor-Ligand HTRF 0 assay for inhibition of IL4 binding to IL4Rα

High-Throughput Screening

Selection outputs were screened in screened in two homogeneoustime-resolved fluorescence (HTRF®) receptor-ligand binding assays, runin parallel to measure inhibitory activity against both human andcynomolgus IL4Rα.

For both assays selection outputs were screened as undiluted or diluted,unpurified bacterial periplasmic extracts containing scFv, prepared in;50 mM MOPS buffer pH7.4, 0.5 mM EDTA and 0.5 M sucrose. All dilutionswere performed in phosphate buffered saline (PBS) containing 0.4 Mpotassium fluoride and 0.1% BSA (assay buffer).

Europium cryptate labelled goat-anti-human Fc antibody, at 3.2 nM (CISBio International 61HFCKLB) was pre-mixed with human IL4Rα/Fc (R and DSystems 604-4R) at 0.5 nM (Premix “A”). XL665-conjugated streptavidin at10 nM (CIS Bio International 611 SAXLB) was pre-mixed with humanbiotinylated IL4 (Pcprotcc with in-house biotinylation) at 4 nM (Pre-Mix“B”).

In parallel, for the cynomolgus assay, europium cryptate labelledgoat-anti-human Fc antibody, at 3.2 nM (CIS Bio International 61HFCKLB)was pre-mixed with cynomolgus IL4RαFc (Isolation/Sf21 expressed) orcynomolgus IL4Rα/Fc HIS6 (Optimisation/HEK-expressed) at 0.5 nM (Premix“A”). XL665-conjugated streptavidin at 10 nM (CIS Bio International611SAXLB) was pre-mixed with cynomolgus biotinylated IL4 (in-house E.coli expressed with biotinylation in-house) at 3 nM (Pre-Mix “B”).

For each assay, 5 p. 1 of Premix “A” was added to a 384 well low volumeassay plate (Costar 3676). 5 μl of unpurified scFv sample was thenadded. This was followed by the addition of 10 μl of Premix “B”.

Non-specific binding was defined using monoclonal mouse IgG2a clone25463 (R and D Systems) at 10 nM final or cynomolgus IL4 at 50 nM final(in-house E. coli expressed) for the human and cynomolgus IL4Rαreceptor-ligand binding assays respectively.

Assay plates were incubated for 4 h at room temperature, prior toreading time resolved fluorescence at 620 nm and 665 nm emissionwavelengths using an EnVision plate reader (Perkin Elmer).

Data was analysed by calculating % Delta F values for each sample. DeltaF was determined according to equation 1.

$\begin{matrix}{{\% \mspace{14mu} {Delta}\mspace{14mu} F} = {\frac{\begin{matrix}{\left( \frac{{sample}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right) -} \\\left( \frac{{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)\end{matrix}}{\left( \frac{{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)} \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

% Delta F values were subsequently used to calculate % specific bindingas described in equation 2.

$\begin{matrix}{{\% \mspace{14mu} {specific}\mspace{14mu} {binding}} = {\frac{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {sample}}{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {binding}\mspace{14mu} {control}} \times 100}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

K_(i) Determination

A dilution series of purified scFv concentrations was prepared todetermine the scFv potency K_(i) values in both human and cynomolgusassays. 5 μl of Premix “A” was added to a 384 well low volume assayplate (Costar 3676). 5 μl of scFv dilution sample was then added. Thiswas followed by the addition of 10 μl of Premix “B”.

Non-specific binding was defined using monoclonal mouse IgG2a clone25463 (R and D Systems) at 10 nM final or cynomolgus IL4 at 50 nM final(in-house E. coli expressed) for the human and cynomolgus IL4Rαreceptor-ligand binding assays respectively.

Assay plates were incubated for 4 h at room temperature, prior toreading time resolved fluorescence at 620 nm and 665 nm emissionwavelengths using an EnVision plate reader (Perkin Elmer).

Data was analysed by calculating % Delta F values for each sample. DeltaF was determined according to equation 1.

$\begin{matrix}{{\% \mspace{14mu} {Delta}\mspace{14mu} F} = {\frac{\begin{matrix}{\left( \frac{{sample}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right) -} \\\left( \frac{{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)\end{matrix}}{\left( \frac{{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {nm}}{620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)} \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

% Delta F values were subsequently used to calculate % specific bindingas described in equation 2.

$\begin{matrix}{{\% \mspace{14mu} {specific}\mspace{14mu} {binding}} = {\frac{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {sample}}{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {binding}\mspace{14mu} {control}} \times 100}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

IC₅₀ values were determined using GraphPad Prism software by curvefitting using a four-parameter logistic equation (Equation 3).

Y=Bottom+(Top−Bottom)/(1+10̂((Log EC50−X)*HillSlope))  Equation 3

X is the logarithm of concentration. Y is specific bindingY starts at Bottom and goes to Top with a sigmoid shape.IC₅₀ values were converted to K, using the Cheng-Prusoff equation asdescribed in equation 4:

K _(i) =IC ₅₀/(1+[L]/K _(d))  Equation 4

Example 3 Reformatting of scFv to IgG2

3.1

Clones were converted from scFv to IgG format by sub-cloning the V_(H)and V_(L) domains into vectors expressing whole antibody heavy and lightchains respectively. The V_(H) domain was cloned into a vector (pEU9.2)containing the human heavy chain constant domains and regulatoryelements to express whole IgG heavy chain in mammalian cells. Similarly,the V_(L) domain was cloned into a vector (pEU4.4) for the expression ofthe human lambda light chain constant domains, with regulatory elementsto express whole IgG light chain in mammalian cells. Vectors for theexpression of heavy chains and light chains were originally described inPeric et al., (Gene 187:9-18, 1997). These vectors have been engineeredsimply by introducing an OriP element. To obtain IgGs, the heavy andlight chain IgG expressing vectors were transfected into EBNA-HEK293mammalian cells (Invitrogen R620-07-). IgGs were expressed and secretedinto the medium. Harvests were pooled and filtered prior topurification. The IgG was purified using Protein A chromatography.Culture supernatants are loaded on a column of appropriate size ofCeramic Protein A (BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium Citrate(pH 3.0) and neutralised by the addition of Tris-HCl (pH 9.0). Theeluted material was buffer exchanged into PBS using Nap10 columns(Amersham, #17-0854-02) and the concentration of IgG was determinedspectrophotometrically using an extinction coefficient based on theamino acid sequence of the IgG (Mach et al., Anal Biochem. 200(1):20-26, 1992). The purified IgG were analysed for aggregation ordegradation using SEC-HPLC and by SDS-PAGE.

3.2 Inhibition of IL-13 and IL-4 Induced Proliferation of TF-1 Cells byIgG

The neutralisation potency of purified IgG preparations against humanIL-13 and IL-4 bioactivity was assessed using TF-1 cell proliferationassay. TF-1 is a human premyeloid cell line established from a patientwith erythroleukemia (Kitamura et al. J. Cell Physiol. 140(2):323-34,1989). The TF-1 cell line is factor dependent for survival andproliferation. TF-1 cells were shown to respond to both human IL-13 andIL-4 (Peprotech, E. coli derived). Inhibition of IL-4 and IL-13dependent proliferation was determined by measuring the reduction inincorporation of tritiated thymidine into the newly synthesized DNA ofdividing cells. A detailed description of the protocol is provided inmaterials and Methods section 3.2.1.

Rc-formatted IgG preparations of Antibody 1 inhibited the IL-13 and IL-4induced proliferation of the TF-1 cells in a concentration dependentmanner. The IC₅₀ geomean for Antibody 1 against IL-13 and IL-4 wascalculated as being 18 nM and 38 nM respectively.

3.2.1 Materials and Methods—Inhibition of IL-4 and IL-13 InducedProliferation of TF-1 Cells by Purified IgG

TF-1 cells (R&D Systems) were maintained according to suppliedprotocols. Assay media comprised RPMI-1640 with GLUTAMAX I (Invitrogen)containing 5% foetal bovine serum (JRH), 1% sodium pyruvate (Sigma),Penicillin/streptomycin (1-2%). Prior to each assay, TF-1 cells werepelleted by centrifugation at 300×g for 5 mins, the media removed byaspiration and the cells re-suspended in assay media. This process wasrepeated twice with cells re-suspended at a final concentration of 2×10⁵cells/ml in assay media. The cells were plated out using 100 μl/well ina 96 well assay plate. Test solutions of IgG (in duplicate) weretitrated to the desired concentration range in assay media. Anirrelevant antibody not directed at IL-4Rα was used as negative control.The assay was set up in a competition format, with 50 uL of eachrecombinant bacterially derived human IL-4 or IL-13 (Peprotech) andappropriate test antibody titrations added sequentially to 100 uL ofcells. A final assay volume of 200 uL/well and a concentration of 18 pM(IL-4) or 400 pM (IL-13) was used in the assay. The concentration ofIL-4 and IL-13 was selected as the dose that at gave approximately 50%of maximal proliferative response. Plates were incubated for 72 hours at37° C. and 5% CO₂ 20 μl of tritiated thymidine (5 μCi/ml) was added toeach assay point and the plates were returned to the incubator forfurther 4 to 5 hours. Cells were harvested on glass fibre filter plates(Perkin Elmer) using a cell harvester. Thymidine incorporation wasdetermined using Packard TopCount microplate liquid scintillationcounter. Data was then analysed using Graphpad Prism software.

Example 4 Antibody Optimisation 4.1 Optimisation of Parent Clone byTargeted Mutagenesis

There are advantages and benefits in the discovery and development of anantibody to human IL4Rα that also exhibits cross-reactivity to theorthologous protein from another species, for example cynomolgus monkey.Such an antibody would facilitate the characterization of suchantibodies with respect to pharmacology and safety in vivo. Potency andaffinity to another species, which is for example less than 10-folddifferent than the human activity may be appropriate for such andevaluation.

To achieve the required species cross-reactivity, the parent antibody(Antibody 1) was optimised for improved affinity to both human andcynomolgus IL4Rα. This was achieved using a targeted mutagenesisapproach with affinity-based phage display selections. For the targetedmutagenesis approach, large scFv-phage libraries derived from theAntibody 1 were created by oligonucleotide-directed mutagenesis of thevariable heavy (V_(H)) and light (V_(L)) chain complementaritydetermining regions 3 (CDR3) using standard molecular biology techniquesas described by Clackson and Lowman (2004) A Practical Approach, 2004.Oxford University Press.

The libraries were subjected to affinity-based phage display selectionsin order to select variants with higher affinity for human andcynomolgus forms of IL-4Rα. The selections were performed essentially asdescribed previously (Thompson. Journal of Molecular Biology. 256:77-88,1996). In brief, the scFv phage particles were incubated with eitherrecombinant biotinylated human or cynomolgus IL-4Rα in solution(bio-huIL-4Rα FLAG HIS or bio-cyno-IL-4RαFc HIS, both in house HEK-EBNAderived). The species of antigen used was alternated at each round ofselection. ScFv-phage bound to antigen were then captured onstreptavidin-coated paramagnetic beads (Dynabcads® M280) following themanufacturer's recommendations. The selected scFv-phage particles werethen rescued as described previously (Osbourn, J K. et al.Immunotechnology, 2(3):181-96, 1996), and the selection process wasrepeated in the presence of decreasing concentrations of eitherbio-huIL-4Rα or bio-cyno-IL-4Rα, alternating the species between roundsof selection (500 nM to 250 pM over 6 rounds).

Upon completion of 6 rounds of selection, the VH and VL randomisedlibraries were recombined to form a single library in which clonescontained randomly paired individually randomised VH and VL sequences.Selections were then continued as previously described in the presenceof decreasing concentrations of either bio-huIL-4Rα or bio-cyno-IL-4Rα(2.5 nM to 0.5 pM over a further 5 rounds), alternating the species ofantigen where appropriate.

4.2 Optimisation of the Antibody by Random Mutagenesis

One of the antibodies (Antibody 20) was further optimised using a randommutagenesis approach to identify key residues within the antibodysequence that may improve binding to human and cynomolgus IL4Rα. LargescFv-phage libraries were generated by the introduction of randommutations throughout the variable regions of the Antibody 20 scFvsequence. This was achieved by two rounds of mutagenesis using aDiversify™ PCR random mutagenesis kit (BD biosciences), following themanufacturers instructions to incorporate on average, 8.1 mutations perkilobasc in the nucleic acid sequence per round of mutagenesis. Theprotocol for the strategy is in accordance with International PatentApplication Publication Number WO2006/072801 (Cambridge AntibodyTechnology). The libraries were subjected to affinity-based phagedisplay selections to select for variants with higher affinity for humanand cynomolgus forms of IL-4Rα.

The selections were performed essentially as described previously(Thompson, Journal of Molecular Biology. 256:77-88, 1996). In brief, thescFv phage particles were incubated with recombinant biotinylated humanor cynomologous IL-4Rα in solution (bio-huIL-4Rα FLAG HIS orbio-cyno-IL-4RαFc HIS, both in house HEK-EBNA derived). The species ofantigen was alternated between human and cynomolgus in order to improveaffinity for the particular species of IL-4Rα accordingly. ScFv-phagebound to antigen were then captured on streptavidin-coated paramagneticbeads (Dynabeads® M280) following the manufacturer's recommendations.The selected scFv-phage particles were then rescued as describedpreviously (Osbourn et al. Immunotechnology, 2(3):181-96, 1996), and theselection process was repeated in the presence of decreasingconcentrations of either bio-huIL-4Rα or bio-cyno-IL-4Rα (20 nM to 1 pMover 4 rounds).

4.3 Identification of Improved Clones from Random Mutagenesis Using aReceptor-Ligand Binding Assay

ScFv from the targeted and random mutagenesis selections were expressedin the bacterial periplasm and screened in two homogeneous time-resolvedfluorescence (HTRF®) receptor-ligand binding assays, run in parallel tomeasure inhibitory activity against both human and cynomolgus IL4Rα. Thedetailed assay method is provided in the Materials and Methods section.2.4. ScFv that showed a significant inhibitory effect in both assays,were subjected to DNA sequencing and scFv with unique sequences wereprepared as purified preparations.

Purified scFv antibody potencies were determined by competing a dilutionseries of the purified scFv preparation against IL4 for binding toIL4Rα. Both human and cynomolgus receptor-ligand assays were performedin parallel as described in the Materials and Methods section 2.4.

Example potency data for scFv for each sample is provided in Table 1.

Ki (nM) Geomean with 95% confidence limit in parentheses scFv Human IL4RCyno IL4R scFv Human IL4R Cyno IL4R Antibody 1 12.0 (8.7, 16.6)Incomplete Antibody 24 49 pM (25, 94) 0.8 (0.5, 1.0) Antibody 2 0.6(0.5, 0.7) 4.3 (2.1, 8.9) Antibody 24PGL 65 pM 0.7 Antibody 3 0.5 48.0Antibody 25 39 pM (20, 79) 1.3 (0.7, 2.7) Antibody 4 0.8 (0.4, 1.8) 6.8(3.4, 13.6) Antibody 26 0.1 1.2 Antibody 5 0.6 (0.1, 2.8) 3.8 (1.8, 8.2)Antibody 27 63 pM 2.8 Antibody 6 0.3 3.5 Antibody 28 94 pM 1.8 Antibody7 1.1 16.5 Antibody 29 61 pM 2.4 Antibody 8 0.4 9.4 Antibody 30 69 pM3.6 Antibody 9 0.1 (0.1, 0.3) 12.9 (9.0, 19) Antibody 31 44 pM (26, 76)2.2 (1.4, 3.4) Antibody 10 0.5 19.6 Antibody 32 75 pM 1.3 Antibody 110.7 26.1 Antibody 33 92 pM 1.4 Antibody 12 0.3 7.2 Antibody 34 56 pM 1.2Antibody 13 0.3 22.2 Antibody 35 71 pM 2.6 Antibody 14 0.6 (0.4, 0.9)27.7 Antibody 36 0.1 5.3 Antibody 15 1.0 (0.8, 1.2) 32.3 Antibody 37 40pM (30, 51) 0.8 (0.7, 1.0) Antibody 16 0.4 (0.2, 1.0) 8.7 Antibody 37 GL61 pM (40, 92) 0.7 (0.5, 1.1) Antibody 17 0.9 43.5 Antibody 38 66 pM 2.7Antibody 18 0.7 56.5 Antibody 39 27 pM 1.6 Antibody 19 0.8 (0.4, 1.6)18.5 Antibody 40 31 pM 6.5 Antibody 20 0.7 (0.6, 0.8) 4.2 (3.4, 5.3)Antibody 41 53 pM 2.7 Antibody 21 31 pM (13, 76) 1.0 (0.5, 1.9) Antibody42 27 pM (16, 49) 2.5 (1.4, 4.5) Antibody 22 0.3 2.6 Antibody 23 0.2 4.8

4.4 Inhibition of IL-13 and IL-4 Induced Proliferation of TF1 Cells byOptimised Clones

Purified scFv antibody potencies were also determined in the TF1proliferation assay. The most potent clones in the TF-1 proliferationassay were converted to IgG as described previously, and were re-testedin the TF-1 proliferation assay. Example potency data for IgG for eachsample is provided in Table 2.

TABLE 2 Example potencies of improved clones when tested in the TF-1cell proliferation assay IC₅₀ (pM) Clone (non-germlined) IL-4 IL-13Antibody 2 41.7 (33.2, 52.3) 171 (68.0, 429) Antibody 4 20.9 (13.5,32.3) 58.1 Antibody 7 12.3 42.7 Antibody 8 7.9 22.4 Antibody 9 8.88(5.94, 13.3) 20.4 (13.9, 29.9) Antibody 10 10.1 25.4 Antibody 11 18.832.7 Antibody 12 18.2 40.7 Antibody 14 3.8 27.2 Antibody 15 2.8 17.8Antibody 16 6.2 19.5 Antibody 19 7.6 22.4 Antibody 20 31.1 (19.9, 48.6)66.1 (34.2, 128)  Antibody 13 15.7 (7.47, 33.1) 24.6 Antibody 21 19.734.7 Antibody 24 12.6 (9.5, 16.7)  30.2 (14.5, 62.8) Antibody 25 9.823.2 Antibody 31 20.2 44.2 Antibody 37 10.4 (7.5, 14.5)  22.1 (11.7,41.8) Antibody 42 20.2 42.7 12B5 * 42.7 (25.9, 70.4) 79.1 (34.7, 180)  *12B5 = Benchmark antibody was made according to the teaching in WO01/92340.

4.5. Germlining

The amino acid sequences of the V_(H) and V_(L) domains of the optimisedanti-IL-4Rα antibodies were aligned to the known human germlinesequences in the VBASE database (MRC Centre For Protein Engineering) andthe closest germline was identified by sequence similarity. For theV_(H) domains of the optimised antibody lineage this wasVh1_DP-7_(1-46). For the VL domains it was Vλ1_DPL5.

Without considering the Vernier residues (Foote & Winter, J Mol. Biol.Mar 20:224(2):487-99, 1992), which were left unchanged, there were 3changes in the frameworks of the V_(H) domains and 2 changes in theV_(L) domains of Antibody 37, all of which were reverted to the closestgermline sequence to identically match human antibodies. Antibody 24 hadone change in the V_(H) domains and 2 changes in the V_(L) domains awayfrom the closest human germline match. Changes at Kabat number 37 in theV_(H) domain and Kabat number 73 in the V_(L) domain were reverted tothat of the closest human germline match. The amino acidchange atposition 87 in the V_(L) domain was left unchanged, to retain potency(Antibody 24PGL). Germlining of these amino acid residues was carriedout using standard site directed mutagenesis techniques with theappropriate mutagenic primers.

The Antibody 37GL scFv sequence was cloned into a cloning vector,transformed into E. coli Top10 cells (F-mcrA Δ(mrr-hsdRMS-msrBC)Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL (StrR)endA1 nupG) and deposited under the Budapest Treaty at NCIMB (Aberdeen,Scotland) on 9 Dec. 2008. The Applicant's clone reference is “Antibody37 GL” and the NCIMB accession number is NCIMB 41600.

Germlined IgG were then re-evaluated in the IL-4 and IL-13 induced TF-1proliferation assay to confirm there had not been a reduction inpotency. Example potencies for germlined (GL) antibodies are provided inTable 3.

TABLE 3 Example potency data for germlined optimised clones whenevaluated in the IL-4 and IL-13 induced TF-1 cell proliferation assayIC₅₀ (pM) Clone (germlined) IL-4 IL-13 Antibody 24 (PGL)  15.3 (6.63,35.1)  55.0 (29.3, 103.1) Antibody 37 (PGL) 14.9 (7.8, 28.6) 41.7 (18.6,93.4) Antibody 37 (FGL) 11.4 (9.8, 13.3) 21.1 (12.4, 35.9)Data are expressed as Geometric mean and 95% confidence intervals

4.6 Selectivity and Species Cross Reactivity of Optimised Antibodies inDELFIA® Epitope Competition Assays

The species cross reactivity and selectivity of antibodies to IL-4Rα andstructurally related molecules; IL13Rα1Fc, IL13Rα2Fc and the commongamma chain (IL-2Rγ), was established using DELFIA® epitope competitionassays. The assay determines relative cross reactivity by measuringinhibition of biotinylated IL-4Rα HIS FLAG (in house HEK-EBNA derived),binding each immobilised anti-IL-4Rα antibody.

Titrations of purified, IL13Rα1Fc, IL13Rα2Fc and the common gamma chain(IL-2Rγ) (all R & D Systems) were tested in each assay to establish thespecificity profile for each structurally related protein, as measuredby IC₅₀ values in the assay.

Titrations of IL-4Rα species including cynomolgus IL-4Rα HIS Fc (inhouse HEK-EBNA derived), human IL-4Rα 175V Fc (AstraZeneca), humanIL-4RαFc and murine IL-4Rα (both R & D Systems) were tested in eachassay to establish the species cross-reactivity of the antibodies.Unbiotinylated human IL-4Rα HIS FLAG was used as a positive control. isHuman and cynomolgus IL-4Rα HIS Fc, along with Human IL-4Rα 175V Fc,produced overlapping inhibition curves with equivocal IC₅₀ values. Noinhibition was observed for murine IL-4Rα or any of the related humanproteins tested. The results demonstrate that Antibody 37GL is crossreactive to cynomolgus 1′-4Rα and human IL-4Rα 175V but does not bind tomurine IL-4Rα or any of the most related human proteins to human IL-4Rα.Details of the protocol are provided in the Materials and Methodssection 4.6.1.

4.6.1 Materials and Methods—DELFIA® Epitope Competition Assays

Purified IgG were adsorbed onto 96-well Maxisorp microlitre plates(Nunc) in PBS at a concentration which gave a significant signal whenbiotinylated human IL-4Rα HIS FLAG was added at approximately itsestimated KD for that particular IgG. Excess IgG was washed away withPBS-Tween (0.1% v/v) and the wells were blocked with PBS-Marvel (3% w/v)for 1 h. A dilution series of each of the following competitors wasprepared in PBS, starting at a concentration of approximately 400-foldthe KD value of the interaction between biotinylated human IL-4Rα andthe respective IgG; human IL-4RαFc (R & D Systems, 604-4R-050), humanIL-4Rcc 175V Fc(AstraZeneca), cynomolgus IL-4Rα HIS Fc (In house),murine IL4Rα Fc(R & D Systems 530-MR-100), Human common gamma chain(sIL-2Rγ) (R & D Systems 384-RG-050-CF), human IL-13Rα1Fc(R & D Systems,146-1R-100), human IL-13Rα2Fc(R & D Systems, 614-1R-100). Unbiotinylatedhuman IL-4Rα HIS FLAG was used as a positive control. To this series, anequal volume of biotinylated recombinant human IL-4Rα at a concentrationof approximately 2-fold the KD was added (resulting in a series startingat a ratio of competitor antigen:biotinylated human IL-Rα ofapproximately 200:1). These mixtures were then transferred onto theblocked IgG and allowed to equilibrate for 1.5 h. Unbound antigen wasremoved by washing with PBS-Tween (0.1% v/v), while the remainingbiotinylated human IL-4Rα was detected by streptavidin-Europium3+conjugate (DELFIA® detection, PerkinElmer). Time-resolved fluorescencewas measured at 620 nm on an EnVision plate reader (PerkinElmer).Fluorescence data was converted to % specific binding (100% wasdetermined from control wells containing biotinylated human IL-4Rα butno competitor, 0% was from wells containing biotinylated human IL-4Rαand a 1000-fold excess of unbiotinylated human IL-4Rα). Resultant datawere analysed using Prism curve is fitting software (Graphpad) todetermine IC₅₀ values.

4.7 Calculation of Affinity Data for Optimised Clones Using Biacore

The binding affinity of purified IgG samples of representative panel ofantibodies to human and cynomolgus IL-4Rα was determined by surfaceplasmon resonance using a BIAcore 2000 biosensor (BIAcore AB)essentially as described by Karlsson et al. (J. Immunol. Methods,145(1-2):229-240, 1991). In brief, Protein G′ (Sigma Aldrich, P4689) wascovalently coupled to the surface of a CM5 sensor chip using standardamine coupling reagents according to manufacturer's instructions(BIAcore). This Protein G′ surface was used to capture a constant amountof purified anti-IL4Rα antibodies or isotype control via the Fc domain.Human or cynomolgus IL-4Rα HIS FLAG prepared in HBS-EP buffer (BIAcoreAB), at a range of concentrations, between 100 nM and 0.2 nM, werepassed over the sensor chip surface. The surface was regenerated using10 mM Glycine, pH 1.75 between each injection of antibody. The resultingsensorgrams were evaluated using BIA evaluation 4.1 software and fittedto 1:1 Langmuir binding model, to provide the relative binding data. Theexperiments were performed over the course of at least three separatedays to calculate an average monovalent affinity. From the data fitsobtained, the affinity of Antibody 37GL to human and cynomolgus IL-4Rαwas determined to be approx 504 pM and 4.4 nM respectively, as reportedin Table 4.

TABLE 4 Example Kinetic Analysis of a representative panel of antibodiesfor binding to human and cyno IL4Rα Human IL4Rα HIS FLAG Cyno IL4Rα HISFLAG Ka (M⁻¹s⁻¹) Kd (s⁻¹) KD (nM) Ka (M⁻¹s⁻¹) Kd (s⁻¹) KD (nM) Antibody2 6.51 × 10⁵ 2.94 × 10⁻³ 4.51 9.67 × 10⁴ 4.59 × 10⁻³ 47.5 Antibody 95.43 × 10⁵ 2.29 × 10⁻⁴ 0.422 1.31 × 10⁵ 1.63 × 10⁻² 124 Antibody 20 6.98× 10⁵ 8.73 × 10⁻⁴ 1.25 1.63 × 10⁵ 4.52 × 10⁻³ 27.7 Antibody 37 6.40 ×10⁵ 1.62 × 10⁻⁴ 0.255 2.43 × 10⁵ 6.88 × 10⁻⁴ 2.97 (0.313, 0.250, 0.203)(2.59, 2.36, 3.96) Antibody 37GL 6.88 × 10⁵ 3.46 × 10⁻⁴ 0.504 2.44 × 10⁵1.08 × 10⁻³ 4.41 (0.533, 0.531, 0.459) (4.67, 4.34, 4.41)

Example 5 Epitope Mapping of IL-4Rα:Antibody Interaction Using ChimericHuman/Mouse IL4Ra Extracellular Domains 5.1 Generation of Whole DomainSwap Chimetic IL-4Rα Molecules.

The binding members of the invention bind strongly to human IL-4Rα butextremely poorly, almost indiscernibly, to mouse IL-4Rα. Using thisproperty, chimeric IL-4Rα molecules were generated for epitope mapping.Whole domain-swap chimeras were created by replacing domain 1 (D1)(M1-E119) or domain 2 (D2) (H120-F229) of human IL-4Rα ectodomain withcorresponding mouse IL-4Rα sequence. Loop swap chimeras were generatedby replacing loop regions known to interact with IL4 (Hage et al., Cell97:271-281, 1999) from human IL-4Rα with corresponding regions frommouse IL-4Rα. An HTRF (homogeneous time resolved fluorescence)competition assay was used to determine chimeras binding to antibody. Inthe assay antibody labelled with Eu³⁺ cryptate interacted with HumanIL-4Rα labelled with biotin. The interaction was detected by a FRET(Fluorescence Resonance Energy Transfer) signal between Eu³⁺ cryptateand XL665 labelled streptavidin (Mathis et al., Clin Chem 41: 1391-1397,1995).

5.1.1 Materials and Methods—Cloning, Expression and Purification ofChimeras

cDNA molecules encoding chimeras of human IL-4Rα extracellular domain(amino acid residues 1-229 NP_(—)000409) and mouse IL-4Rα extracellulardomain (amino acid residues 1-230 NP_(—)001008700) were synthesised byprimer extension PCR cloning and cloned into pDONR221 (Invitrogen Cat.No. 12536-017). The cDNA fragments coding for the IL-4Rα extracellulardomain chimeras were then transferred to mammalian expression vectorpDEST12.2 (Invitrogen) using LR Gateway Clonase II enzyme according tothe manufacturer's instructions (Invitrogen Cat. No. 12538-120). ThepDEST12.2 vector had been modified to contain a FLAG 10xhis tag(DYKDDDDKAAHHHHHHHHHH; e.g. see SEQ TD NO: 460, positions 252-261)in-frame with the inserted gene of interest, and also by insertion ofthe oriP origin of replication from the pCEP4 vector (Invitrogen cat.no. VO44-50) allowing episomal plasmid replication upon transfectioninto cell lines expressing the EBNA-1 gene product (such as HEK293-EBNAcells). Expressed protein in HEK293-EBNA supernatant was purified usingNi-NTA affinity chromatography (Histrap HP column (GE Healthcare Cat.No. 17-5248-02)) followed by Size Exclusion chromatography (Superdex 200column (GE Healthcare Cat. No. 17-1069-01)).

The sequence of human IL-4Rα extracellular domain (positions 1-229; andsame in NP_(—)000409), vector encoded sequence (positions 230-241), FLAGtag (positions 242-249) and 10xhis tag (positions 252-261) is disclosedin SEQ ID NO: 460. The sequence of murine IL-4Rα extracellular domain(positions 1-230; and same in NP_(—)001008700), vector encoded sequence(positions 233-242), FLAG tag (positions 243-250) and 10xhis tag(positions 253-262) is disclosed in SEQ ID NO: 461.

5.1.2 Binding of Antibody to IL-4Rα Chimeras

Antibody was cryptate labelled with Eu³⁺ Cryptate labelling kitaccording to the manufacturer's instructions (CisBio International CatNo. 62EUSPEA) and IL-4Rα/Fc (R&D systems Cat. No. 604-4R-050) was Biotinlabelled with EZ Link NHS-Biotin according to the manufacturer'sinstructions (Perbio Cat No. 20217). Assay conditions were 0.4 nMCryptate labelled antibody, 0.25 nM biotin labelled IL-4Rα/Fc, 2.5 nMstreptavidin XL665 (CisBio International Cat. No. 611SAXLB) in 1×DPBS,0.1% BSA, 0.4M potassium fluoride in a total volume of 20 μl in a 384well microtitre plate (Greiner). To the assay a dilution series (from1000 nM to 0.017 nM) of test proteins was added and the assay incubatedovernight at 4° C. FRET signal was detected using a PerkinElmer EnVisionplate reader using a 320 nm excitation filter and 620 nm and 665 nmemission filters. Results were calculated from the 665/620 ratio as apercentage of specific binding (signal with no competitor antigen).Results were analysed with Prism (GraphPad Software) using the sigmoidaldose response model.

5.2 Results

Antibody binding of chimeric molecules was tested in an HTRF(Homogeneous Time Resolved Fluorescence) competition assay. Moleculeswhich bound antibody at the same paratope as human IL-4Rα inhibited thebinding interaction, leading to a reduction in signal. From inhibitioncurves IC₅₀ values for human IL-4Rα, mouse IL-4Rα and chimeric moleculeswere calculated (Table 5). If a molecule did not fully inhibit bindingthe percentage inhibition seen at the highest concentration wascalculated. Chimeras that gave similar IC₅₀ values to native humanIL-4Rα still contained the epitope. Chimeras, which did not fullyinhibit IL-4Rα binding to antibody, or showed an increased IC₅₀ value,did not contain the full epitope. These data enabled the localisation ofthe antibody epitope.

TABLE 5 IC₅₀ (in nM) of chimeric IL-4Rα molecules competing againsthuman IL-4Rα binding to antibody. Sequence from Sequence from IC₅₀ (nM)in Chimera NP_000409 NP_001008700 competition assay HuIL-4Rα M1-F2290.673 MoIL-4Rα M1-L230 45% inhibition* HuD1MoD2IL- M1-E119 N121-L230 3.44Rα MoD1HuD2IL- H120-F229 M1-G120 34% inhibition* 4Rα MoLoop1HuIL-M1-Y38; S42-F229 I39-T41 1.9 4Rα MoLoop2HuIL- M1-L64; T73-F229 M65-L7323 4Rα MoLoop3HuIL- M1-L88; Y99-F229 E90-R99 58% inhibition* 4RαMoLoop4HuIL- M1-W143; N151-F229 N145-N151 0.663 4Rα MoLoop5HuIL-M1-W204; T211-F229 S206-G211 1.4 4Rα AllMoLoopsHuIL- M1-Y38; S42-L88;Y99- I39-T41; M65-173; 41% inhibition* 4Rα W143; N151-W204; E90-R99;N145- T211-F229 N151; S206-G211 Chimeras composed of amino acid sequencefrom human (NP_000409) and mouse (NP_001008700) IL-4Rα were tested forthe ability to compete with human IL-4Rα in binding antibody. IC₅₀values were calculated where a complete competition curve was obtained.*Where the chimera failed to completely inhibit human IL-4Rα binding toantibody the percentage inhibition seen at the highest concentration ofchimera (1000 nM) is shown.

The binding of chimeric human/mouse IL-4Rα chimeras has enabled thelocalisation of the human IL-4Rα epitope bound by antibody. Whole domainswap chimeras localised the epitope to D1 of human IL-4Rα. (residuesM1-E119) as a human D1-mouse D2 chimera was able to compete with humanIL-4Rα whereas a mouse D1-human D2 chimera failed to completely inhibitHuman IL-4Rα binding (Table 5). The epitope is almost entirely composedof loop regions since the AllMoLoops chimera and MoIL-4Rα show verysimilar percentage inhibition (Table 5).

Human IL-4Rα contains five loop regions, which are in close proximity toIL4 in a crystal structure (Hage et al., Cell 97:271-281, 1999). Loopswap chimeras enabled the localisation of the human IL-4Rα epitope to amajor component in loop 3 (residues L89-N98) and a minor component inloop 2 (residues V65-H72). Chimeras without human loop 3 failed toinhibit human IL-4Rα binding to antibody and chimeras without loop 2gave a 100 fold higher IC₅₀ than human IL-4Rα (Table 5). Consistent withthe domain swap data both loop2 and loop3 are located in D1 (Hage etal., Cell 97:271-281, 1999).

From these data the antibody epitope was located to a discontinuousepitope of 18 amino acids in two loop regions of human IL-4Rα; V65-H72and L89-N98.

5.3 Further Localisation of the IL-4Rα Epitope of Antibody using Mutantsof MoLoop2Hu IL-4Rα and MoLoop3HuIL-4Rα

To localise important residues of the antibody epitope of human IL-4Rαmutants of MoLoop2HuIL-4Rα and MoLoop3HuIL-4Rα were generated and testedfor activity in an HTRF competition assay. MoLoop2HuIL-4Rα (Table 5) wasmutated to convert mouse residues back to human. When epitope-importantresidues were mutated, the IC₅₀ values were lower than that ofMoLoop2HuIL-4Rα (Table 6). Three mutants (constructs EM18, EM22 andEM23) gave similar IC₅₀ values to MoLoop2HuIL-4Rα (Table 6). In additionwhen mouse residues N72 is and L73 are placed to human IL-4Rα theresultant chimeric molecule is able to strongly inhibit the humanIL-4Rα/antibody interaction (Table 6). These data suggest that humanIL-4Rα residues V65, A71 and H72 are not important parts of the antibodyepitope. The remaining three mouse residues of loop 2 (F67, E68, F69)correspond to two human residues (L67, L68). In MoLoop2HuIL-4Rα mutantswhere any of the two mouse phenylalanine residues is replaced with ahuman lysine residue the IC₅₀ is reduced (Table 6). When mouse E68 isremoved from MoLoop2HuIL-4Rα the IC₅₀ is also reduced suggesting thatacidic glutamic acid residue of mouse loop2 is blocking part of theantibody epitope. In addition when all three mouse residues (F67, E68,F69) are placed into human IL-4Rα the chimera only weakly inhibits humanIL-4Rα/antibody interaction (Table 6). These data show human residuesL67 and L68 are part of the antibody binding epitope of human IL-4Rα.

TABLE 6 IC₅₀ (in nM) of chimeric IL-4Rα molecules competing againsthuman IL-4Rα binding to antibody. Construct name Loop2 sequence IC₅₀(nM) in competition assay Human IL-4Rα

0.673 MoLoop2HuIL-4Rα MfFEFscNL 25 EM18

20 EM19

4.5 EM20 MfF-FseNL 3.8 EM21

5.7 EM22

19 EM23

56 EM24

13 EM25

0.448 Chimeras composed of amino acids M1-L64 and T73-F229 from humanIL-4Rα (NP_000409) with different loop2 regions were tested for theability to compete with human IL-4Rα in binding antibody. Conservedresidues between human and mouse are shown in lowercase, human residuesdifferent from mouse residues are shown in uppercase bold italics andmouse residues different from human residues are shown in uppercaseplain text. IC₅₀ values were calculated where a complete competitioncurve was obtained.

For loop 3 mutants in human IL-4Rα were constructed where individualresidues were changed to mouse residues (Table 7). A lower IC₅₀ thanhuman IL-4Rα is seen when epitope-important residues arc mutated.

Two mutants had drastically reduced ability to block the humanIL-4Rα/antibody interaction showing that they are part of the epitope.When human IL-4Rα D92 is mutated to R (in mutant EM03) the mutantprotein is unable to block the IL-4Rα/antibody interaction at 1000 nM.When human IL-4Rα V93 is mutated to P (in mutant EM04) the IC₅₀ value is20 fold higher than human IL-4Rα (Table 7). In comparison the humanIL-4Rα mutants L89E, D91N, S95Q, A96V and N98R (chimeras EM01, EM02,EM05, EM06 and EM07) all gave similar IC₅₀ values to human IL-4Rα (Table3). These data suggest that the antibody epitope includes human IL-4RαD92 and V93 and that D92 is the most important residue in the humanIL-4Rα/antibody interaction.

TABLE 7 IC₅₀ (in nM) of chimeric IL-4Rα molecules competing againsthuman IL-4Rα binding to antibody. Construct name Loop3 sequence IC₅₀(nM) in competition assay Human IL-4Rα

0.673 MoLoop3HuIL-4Rα EmNRPvQSdR 45% inhibition* EM01

0.71 EM02

2.25 EM03

29% inhibition* EM04

15 EM05

1.65 EM06

3.25 EM07

1.41 Chimeras composed of amino acids M1-L88 and Y99-F229 from humanIL-4Rα (NP_000409) with different loop3 regions were tested for theability to compete with human IL-4Rα in binding antibody. Conservedresidues between human and mouse are shown in lowercase, human residuesdifferent from mouse residues are shown in uppercase bold italics andmouse residues different from human residues are shown in uppercaseplain text. IC₅₀ values were calculated where a complete competitioncurve was obtained. *Where the chimera failed to completely inhibithuman IL-4Rα binding to antibody the percentage inhibition seen at thehighest concentration of chimera (1000 nM) is shown.

Domain swapping and mutagenesis has localised the antibody epitope toloops 2 (residues 65 to 72) and 3 (residues 89-98) of human IL-4Rα. Theepitope can be further localised to amino acid residues L67 and L68 ofloop 2 and D92 and V93 of loop3 (see SEQ ID NO: 454 for location ofresidues 67, 68, 92 and 93).

The antibodies of the invention are also cross-reactive with cynomologusmonkey IL-4Rα. Of interest is the fact that the epitope residues D92 andV93 are also present in cynomologus monkey IL-4Rα.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference in to thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Sequences

Sequences of binding members of the invention are shown in the appendedsequence listing, in which SEQ ID Nos correspond as follows:

Sequence Description 1 Antibody 1 VH DNA 2 Antibody 1 VH PRT 3 Antibody1 CDR1 PRT 4 Antibody 1 CDR2 PRT 5 Antibody 1 CDR3 PRT 6 Antibody 1 VLDNA 7 Antibody 1 VL PRT 8 Antibody 1 CDR1 PRT 9 Antibody 1 CDR2 PRT 10Antibody 1 CDR3 PRT 11 Antibody 2 VH DNA 12 Antibody 2 VH PRT 13Antibody 2 CDR1 PRT 14 Antibody 2 CDR2 PRT 15 Antibody 2 CDR3 PRT 16Antibody 2 VL DNA 17 Antibody 2 VL PRT 18 Antibody 2 CDR1 PRT 19Antibody 2 CDR2 PRT 20 Antibody 2 CDR3 PRT 21 Antibody 3 VH DNA 22Antibody 3 VH PRT 23 Antibody 3 CDR1 PRT 24 Antibody 3 CDR2 PRT 25Antibody 3 CDR3 PRT 26 Antibody 3 VL DNA 27 Antibody 3 VL PRT 28Antibody 3 CDR1 PRT 29 Antibody 3 DR2 PRT 30 Antibody 3 CDR3 PRT 31Antibody 4 VH DNA 32 Antibody 4 VH PRT 33 Antibody 4 CDR1 PRT 34Antibody 4 CDR2 PRT 35 Antibody 4 CDR3 PRT 36 Antibody 4 VL DNA 37Antibody 4 VL PRT 38 Antibody 4 CDR1 PRT 39 Antibody 4 CDR2 PRT 40Antibody 4 CDR3 PRT 41 Antibody 5 VH DNA 42 Antibody 5 VH PRT 43Antibody 5 CDR1 PRT 44 Antibody 5 CDR2 PRT 45 Antibody 5 CDR3 PRT 46Antibody 5 VL DNA 47 Antibody 5 VL PRT 48 Antibody 5 CDR1 PRT 49Antibody 5 CDR2 PRT 50 Antibody 5 CDR3 PRT 51 Antibody 6 VH DNA 52Antibody 6 VH PRT 53 Antibody 6 CDR1 PRT 54 Antibody 6 CDR2 PRT 55Antibody 6 CDR3 PRT 56 Antibody 6 VL DNA 57 Antibody 6 VL PRT 58Antibody 6 CDR1 PRT 59 Antibody 6 CDR2 PRT 60 Antibody 6 CDR3 PRT 61Antibody 7 VH DNA 62 Antibody 7 VH PRT 63 Antibody 7 CDR1 PRT 64Antibody 7 CDR2 PRT 65 Antibody 7 CDR3 PRT 66 Antibody 7 VL DNA 67Antibody 7 VL PRT 68 Antibody 7 CDR1 PRT 69 Antibody 7 CDR2 PRT 70Antibody 7 CDR3 PRT 71 Antibody 8 VH DNA 72 Antibody 8 VH PRT 73Antibody 8 CDR1 PRT 74 Antibody 8 CDR2 PRT 75 Antibody 8 CDR3 PRT 76Antibody 8 VL DNA 77 Antibody 8 VL PRT 78 Antibody 8 CDR1 PRT 79Antibody 8 CDR2 PRT 80 Antibody 8 CDR3 PRT 81 Antibody 9 VH DNA 82Antibody 9 VH PRT 83 Antibody 9 CDR1 PRT 84 Antibody 9 CDR2 PRT 85Antibody 9 CDR3 PRT 86 Antibody 9 VL DNA 87 Antibody 9 VL PRT 88Antibody 9 CDR1 PRT 89 Antibody 9 CDR2 PRT 90 Antibody 9 CDR3 PRT 91Antibody 10 VH DNA 92 Antibody 10 VH PRT 93 Antibody 10 CDR1 PRT 94Antibody 10 CDR2 PRT 95 Antibody 10 CDR3 PRT 96 Antibody 10 VL DNA 97Antibody 10 VL PRT 98 Antibody 10 CDR1 PRT 99 Antibody 10 CDR2 PRT 100Antibody 10 CDR3 PRT 101 Antibody 11 VH DNA 102 Antibody 11 VH PRT 103Antibody 11 CDR1 PRT 104 Antibody 11 CDR2 PRT 105 Antibody 11 CDR3 PRT106 Antibody 11 VL DNA 107 Antibody 11 VL PRT 108 Antibody 11 CDR1 PRT109 Antibody 11 CDR2 PRT 110 Antibody 11 CDR3 PRT 111 Antibody 12 VH DNA112 Antibody 12 VH PRT 113 Antibody 12 CDR1 PRT 114 Antibody 12 CDR2 PRT115 Antibody 12 CDR3 PRT 116 Antibody 12 VL DNA 117 Antibody 12 VL PRT118 Antibody 12 CDR1 PRT 119 Antibody 12 CDR2 PRT 120 Antibody 12 CDR3PRT 121 Antibody 13 VH DNA 122 Antibody 13 VH PRT 123 Antibody 13 CDR1PRT 124 Antibody 13 CDR2 PRT 125 Antibody 13 CDR3 PRT 126 Antibody 13 VLDNA 127 Antibody 13 VL PRT 128 Antibody 13 CDR1 PRT 129 Antibody 13 CDR2PRT 130 Antibody 13 CDR3 PRT 131 Antibody 14 VH DNA 132 Antibody 14 VHPRT 133 Antibody 14 CDR1 PRT 134 Antibody 14 CDR2 PRT 135 Antibody 14CDR3 PRT 136 Antibody 14 VL DNA 137 Antibody 14 VL PRT 138 Antibody 14CDR1 PRT 139 Antibody 14 CDR2 PRT 140 Antibody 14 CDR3 PRT 141 Antibody15 VH DNA 142 Antibody 15 VH PRT 143 Antibody 15 CDR1 PRT 144 Antibody15 CDR2 PRT 145 Antibody 15 CDR3 PRT 146 Antibody 15 VL DNA 147 Antibody15 VL PRT 148 Antibody 15 CDR1 PRT 149 Antibody 15 CDR2 PRT 150 Antibody15 CDR3 PRT 151 Antibody 16 VH DNA 152 Antibody 16 VH PRT 153 Antibody16 CDR1 PRT 154 Antibody 16 CDR2 PRT 155 Antibody 16 CDR3 PRT 156Antibody 16 VL DNA 157 Antibody 16 VL PRT 158 Antibody 16 CDR1 PRT 159Antibody 16 CDR2 PRT 160 Antibody 16 CDR3 PRT 161 Antibody 17 VH DNA 162Antibody 17 VH PRT 163 Antibody 17 CDR1 PRT 164 Antibody 17 CDR2 PRT 165Antibody 17 CDR3 PRT 166 Antibody 17 VL DNA 167 Antibody 17 VL PRT 168Antibody 17 CDR1 PRT 169 Antibody 17 CDR2 PRT 170 Antibody 17 CDR3 PRT171 Antibody 18 VH DNA 172 Antibody 18 VH PRT 173 Antibody 18 CDR1 PRT174 Antibody 18 CDR2 PRT 175 Antibody 18 CDR3 PRT 176 Antibody 18 VL DNA177 Antibody 18 VL PRT 178 Antibody 18 CDR1 PRT 179 Antibody 18 CDR2 PRT180 Antibody 18 CDR3 PRT 181 Antibody 19 VH DNA 182 Antibody 19 VH PRT183 Antibody 19 CDR1 PRT 184 Antibody 19 CDR2 PRT 185 Antibody 19 CDR3PRT 186 Antibody 19 VL DNA 187 Antibody 19 VL PRT 188 Antibody 19 CDR1PRT 189 Antibody 19 CDR2 PRT 190 Antibody 19 CDR3 PRT 191 Antibody 20 VHDNA 192 Antibody 20 VH PRT 193 Antibody 20 CDR1 PRT 194 Antibody 20 CDR2PRT 195 Antibody 20 CDR3 PRT 196 Antibody 20 VL DNA 197 Antibody 20 VLPRT 198 Antibody 20 CDR1 PRT 199 Antibody 20 CDR2 PRT 200 Antibody 20CDR3 PRT 201 Antibody 21 VH DNA 202 Antibody 21 VH PRT 203 Antibody 21CDR1 PRT 204 Antibody 21 CDR2 PRT 205 Antibody 21 CDR3 PRT 206 Antibody21 VL DNA 207 Antibody 21 VL PRT 208 Antibody 21 CDR1 PRT 209 Antibody21 CDR2 PRT 210 Antibody 21 CDR3 PRT 211 Antibody 22 VH DNA 212 Antibody22 VH PRT 213 Antibody 22 CDR1 PRT 214 Antibody 22 CDR2 PRT 215 Antibody22 CDR3 PRT 216 Antibody 22 VL DNA 217 Antibody 22 VL PRT 218 Antibody22 CDR1 PRT 219 Antibody 22 CDR2 PRT 220 Antibody 22 CDR3 PRT 221Antibody 23 VH DNA 222 Antibody 23 VH PRT 223 Antibody 23 CDR1 PRT 224Antibody 23 CDR2 PRT 225 Antibody 23 CDR3 PRT 226 Antibody 23 VL DNA 227Antibody 23 VL PRT 228 Antibody 23 CDR1 PRT 229 Antibody 23 CDR2 PRT 230Antibody 23 CDR3 PRT 231 Antibody 24 VH DNA 232 Antibody 24 VH PRT 233Antibody 24 CDR1 PRT 234 Antibody 24 CDR2 PRT 235 Antibody 24 CDR3 PRT236 Antibody 24 VL DNA 237 Antibody 24 VL PRT 238 Antibody 24 CDR1 PRT239 Antibody 24 CDR2 PRT 240 Antibody 24 CDR3 PRT 241 Antibody 25 VH DNA242 Antibody 25 VH PRT 243 Antibody 25 CDR1 PRT 244 Antibody 25 CDR2 PRT245 Antibody 25 CDR3 PRT 246 Antibody 25 VL DNA 247 Antibody 25 VL PRT248 Antibody 25 CDR1 PRT 249 Antibody 25 CDR2 PRT 250 Antibody 25 CDR3PRT 251 Antibody 26 VH DNA 252 Antibody 26 VH PRT 253 Antibody 26 CDR1PRT 254 Antibody 26 CDR2 PRT 255 Antibody 26 CDR3 PRT 256 Antibody 26 VLDNA 257 Antibody 26 VL PRT 258 Antibody 26 CDR1 PRT 259 Antibody 26 CDR2PRT 260 Antibody 26 CDR3 PRT 261 Antibody 27 VH DNA 262 Antibody 27 VHPRT 263 Antibody 27 CDR1 PRT 264 Antibody 27 CDR2 PRT 265 Antibody 27CDR3 PRT 266 Antibody 27 VL DNA 267 Antibody 27 VL PRT 268 Antibody 27CDR1 PRT 269 Antibody 27 CDR2 PRT 270 Antibody 27 CDR3 PRT 271 Antibody28 VH DNA 272 Antibody 28 VH PRT 273 Antibody 28 CDR1 PRT 274 Antibody28 CDR2 PRT 275 Antibody 28 CDR3 PRT 276 Antibody 28 VL DNA 277 Antibody28 VL PRT 278 Antibody 28 CDR1 PRT 279 Antibody 28 CDR2 PRT 280 Antibody28 CDR3 PRT 281 Antibody 29 VH DNA 282 Antibody 29 VH PRT 283 Antibody29 CDR1 PRT 284 Antibody 29 CDR2 PRT 285 Antibody 29 CDR3 PRT 286Antibody 29 VL DNA 287 Antibody 29 VL PRT 288 Antibody 29 CDR1 PRT 289Antibody 29 CDR2 PRT 290 Antibody 29 CDR3 PRT 291 Antibody 30 VH DNA 292Antibody 30 VH PRT 293 Antibody 30 CDR1 PRT 294 Antibody 30 CDR2 PRT 295Antibody 30 CDR3 PRT 296 Antibody 30 VL DNA 297 Antibody 30 VL PRT 298Antibody 30 CDR1 PRT 299 Antibody 30 CDR2 PRT 300 Antibody 30 CDR3 PRT301 Antibody 31 VH DNA 302 Antibody 31 VH PRT 303 Antibody 31 CDR1 PRT304 Antibody 31 CDR2 PRT 305 Antibody 31 CDR3 PRT 306 Antibody 31 VL DNA307 Antibody 31 VL PRT 308 Antibody 31 CDR1 PRT 309 Antibody 31 CDR2 PRT310 Antibody 31 CDR3 PRT 311 Antibody 32 VH DNA 312 Antibody 32 VH PRT313 Antibody 32 CDR1 PRT 314 Antibody 32 CDR2 PRT 315 Antibody 32 CDR3PRT 316 Antibody 32 VL DNA 317 Antibody 32 VL PRT 318 Antibody 32 CDR1PRT 319 Antibody 32 CDR2 PRT 320 Antibody 32 CDR3 PRT 321 Antibody 33 VHDNA 322 Antibody 33 VH PRT 323 Antibody 33 CDR1 PRT 324 Antibody 33 CDR2PRT 325 Antibody 33 CDR3 PRT 326 Antibody 33 VL DNA 327 Antibody 33 VLPRT 328 Antibody 33 CDR1 PRT 329 Antibody 33 CDR2 PRT 330 Antibody 33CDR3 PRT 331 Antibody 34 VH DNA 332 Antibody 34 VH PRT 333 Antibody 34CDR1 PRT 334 Antibody 34 CDR2 PRT 335 Antibody 34 CDR3 PRT 336 Antibody34 VL DNA 337 Antibody 34 VL PRT 338 Antibody 34 CDR1 PRT 339 Antibody34 CDR2 PRT 340 Antibody 34 CDR3 PRT 341 Antibody 35 VH DNA 342 Antibody35 VH PRT 343 Antibody 35 CDR1 PRT 344 Antibody 35 CDR2 PRT 345 Antibody35 CDR3 PRT 346 Antibody 35 VL DNA 347 Antibody 35 VL PRT 348 Antibody35 CDR1 PRT 349 Antibody 35 CDR2 PRT 350 Antibody 35 CDR3 PRT 351Antibody 36 VH DNA 352 Antibody 36 VH PRT 353 Antibody 36 CDR1 PRT 354Antibody 36 CDR2 PRT 355 Antibody 36 CDR3 PRT 356 Antibody 36 VL DNA 357Antibody 36 VL PRT 358 Antibody 36 CDR1 PRT 359 Antibody 36 CDR2 PRT 360Antibody 36 CDR3 PRT 361 Antibody 37 VH DNA 362 Antibody 37 VH PRT 363Antibody 37 CDR1 PRT 364 Antibody 37 CDR2 PRT 365 Antibody 37 CDR3 PRT366 Antibody 37 VL DNA 367 Antibody 37 VL PRT 368 Antibody 37 CDR1 PRT369 Antibody 37 CDR2 PRT 370 Antibody 37 CDR3 PRT 371 Antibody 38 VH DNA372 Antibody 38 VH PRT 373 Antibody 38 CDR1 PRT 374 Antibody 38 CDR2 PRT375 Antibody 38 CDR3 PRT 376 Antibody 38 VL DNA 377 Antibody 38 VL PRT378 Antibody 38 CDR1 PRT 379 Antibody 38 CDR2 PRT 380 Antibody 38 CDR3PRT 381 Antibody 39 VH DNA 382 Antibody 39 VH PRT 383 Antibody 39 CDR1PRT 384 Antibody 39 CDR2 PRT 385 Antibody 39 CDR3 PRT 386 Antibody 39 VLDNA 387 Antibody 39 VL PRT 388 Antibody 39 CDR1 PRT 389 Antibody 39 CDR2PRT 390 Antibody 39 CDR3 PRT 391 Antibody 40 VH DNA 392 Antibody 40 VHPRT 393 Antibody 40 CDR1 PRT 394 Antibody 40 CDR2 PRT 395 Antibody 40CDR3 PRT 396 Antibody 40 VL DNA 397 Antibody 40 VL PRT 398 Antibody 40CDR1 PRT 399 Antibody 40 CDR2 PRT 400 Antibody 40 CDR3 PRT 401 Antibody41 VH DNA 402 Antibody 41 VH PRT 403 Antibody 41 CDR1 PRT 404 Antibody41 CDR2 PRT 405 Antibody 41 CDR3 PRT 406 Antibody 41 VL DNA 407 Antibody41 VL PRT 408 Antibody 41 CDR1 PRT 409 Antibody 41 CDR2 PRT 410 Antibody41 CDR3 PRT 411 Antibody 42 VH DNA 412 Antibody 42 VII PRT 413 Antibody42 CDR1 PRT 414 Antibody 42 CDR2 PRT 415 Antibody 42 CDR3 PRT 416Antibody 42VL DNA 417 Antibody 42 VL PRT 418 Antibody 42 CDR1 PRT 419Antibody 42 CDR2 PRT 420 Antibody 42 CDR3 PRT 421 Antibody 24 PGLVH DNA422 Antibody 24 PGLVH PRT 423 Antibody 24 PGLCDR1 PRT 424 Antibody 24PGLCDR2 PRT 425 Antibody 24 PGLCDR3 PRT 426 Antibody 24 PGLVL DNA 427Antibody 24 PGLVL PRT 428 Antibody 24 PGLCDR1 PRT 429 Antibody 24PGLCDR2 PRT 430 Antibody 24 PGLCDR3 PRT 431 Antibody 24 GLVH DNA 432Antibody 24 GLVH PRT 433 Antibody 24 GLCDR1 PRT 434 Antibody 24 GLCDR2PRT 435 Antibody 24 GLCDR3 PRT 436 Antibody 24 GLVL DNA 437 Antibody 24GLVL PRT 438 Antibody 24 GLCDR1 PRT 439 Antibody 24 GLCDR2 PRT 440Antibody 24 GLCDR3 PRT 441 Antibody 37 GLVH DNA 442 Antibody 37 GLVH PRT443 Antibody 37 GLCDR1 PRT 444 Antibody 37 GLCDR2 PRT 445 Antibody 37GLCDR3 PRT 446 Antibody 37 GLVL DNA 447 Antibody 37 GLVL PRT 448Antibody 37 GLCDR1 PRT 449 Antibody 37 GLCDR2 PRT 450 Antibody 37 GLCDR3PRT 451 Cyno IL4R primer 1 452 Cyno IL4R primer 2 453 I75Voligonucleotide mutation primer 454 Human IL-4Rα/Fc protein 455Cynomolgus monkey IL-4Rα cDNA nucleotide 456 Cynomolgus monkey IL-4Rα/FccDNA nucleotide 457 Cynomolgus monkey IL-4Rα/Fc cDNA protein 458 HumanI75V IL4-Rα/Fc cDNA nucleotide sequence 459 Human I75V IL4-Rα/Fc protein460 Human IL4-Rα/FLAG/HIS tag amino acid sequence 461 MurineIL4-Rα/FLAG/HIS tag amino acid sequence

1-19. (canceled)
 20. A method of treating a disorder associated withaberrant IL-4Rα expression and/or IL-4Rα activity in an individual,comprising administering a therapeutically effective amount of anisolated antibody or fragment thereof that specifically binds humaninterleukin-4 receptor alpha (hIL-4Rα), wherein the isolated antibody orfragment thereof comprises a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3, wherein the set of CDRs has 10 or fewer amino acidsubstitutions from a reference set of CDRs in which: HCDR1 has aminoacid sequence SEQ ID NO: 193; HCDR2 has amino acid sequence SEQ ID NO:194; HCDR3 has amino acid sequence SEQ ID NO: 195; LCDR1 has amino acidsequence SEQ ID NO: 198; LCDR2 has amino acid sequence SEQ ID NO: 199;and LCDR3 has amino acid sequence SEQ ID NO:
 200. 21. The methodaccording to claim 20, wherein the disorder is selected from the groupconsisting of allergy, asthma, bronchitis, COPD, inflammatory boweldisease, fibrotic conditions, allergy, transplant rejection,delayed-type hypersensitivity or contact hypersensitivity reactions. 22.The method according to claim 20, wherein the disorder is asthma. 23.The method according to claim 20, wherein the isolated antibody orfragment thereof is an IgG1, IgG2 or IgG4 molecule.
 24. The methodaccording to claim 20 wherein the amino acid substitutions comprise oneor more substitutions as shown in FIGS. 3 and
 4. 25. The methodaccording to claim 20 wherein the amino acid substitutions comprise anamino acid substitution at one or more of the following residues withinthe CDRs, using the standard numbering of Kabat: 53, 57, in HCDR2; 97,98, 99, 101, 102 in HCDR3; 27, 27A, 27B, 31 in LCDR1; 56 in LCDR2; or92, 93, 94, 95, 95A 95B, 95C, 96, 97 in LCDR3.
 26. The method accordingto claim 20 wherein the antibody or fragment thereof further comprisesone or more amino acid substitutions at the following residues withinthe framework regions, using the standard numbering of Kabat: 11, 12 inHFW1; 37, 48 in HFW2; 68, 84, 85 in HFW3; 105, 108, 113 in HFW4; 1, 2,3, 9 in LFW1; 38, 42 in LFW2; or 58, 65, 66, 70, 74, 85, 87 in LFW3. 27.A method of treating a disorder associated with aberrant IL-4Rαexpression and/or IL-4Rα activity in an individual, comprisingadministering a therapeutically effective amount of an isolated antibodyor fragment thereof that specifically binds human interleukin-4 receptoralpha (hIL-4Rα), wherein: (i) the HCDR1 has amino acid sequence SEQ IDNO: 363; the HCDR2 has amino acid sequence SEQ ID NO: 364; the HCDR3 hasamino acid sequence SEQ ID NO: 365; the LCDR1 has amino acid sequenceSEQ ID NO: 368; the LCDR2 has amino acid sequence SEQ ID NO: 369; andthe LCDR3 has amino acid sequence SEQ ID NO: 370; or (ii) the HCDR1 hasamino acid sequence SEQ ID NO: 233; the HCDR2 has amino acid sequenceSEQ ID NO: 234; the HCDR3 has amino acid sequence SEQ ID NO: 235; theLCDR1 has amino acid sequence SEQ ID NO: 238; the LCDR2 has amino acidsequence SEQ ID NO: 239; and the LCDR3 has amino acid sequence SEQ IDNO:
 240. 28. The method according to claim 20 or 27 wherein the antibodyor fragment thereof comprises an antibody VH domain and an antibody VLdomain, wherein the VH domain comprises HCDR1, HCDR2, HCDR3 and a firstframework and the VL domain comprises LCDR1, LCDR2, LCDR3 and a secondframework.
 29. The method according to claim 27 wherein the isolatedantibody or fragment thereof is an IgG1, IgG2 or IgG4 molecule.
 30. Amethod of treating a disorder associated with aberrant IL-4Rα expressionand/or IL-4Rα activity in an individual, comprising administering atherapeutically effective amount of an isolated antibody or fragmentthereof that specifically binds human interleukin-4 receptor alpha(hIL-4Rα), wherein the isolated antibody or fragment thereof comprises aVH and a VL domain wherein: (i) the VH domain has amino acid sequenceSEQ ID NO: 192 and the VL domain has amino acid sequence SEQ ID NO: 197;(ii) the VH domain has amino acid sequence SEQ ID NO: 362 and the VLdomain has amino acid sequence SEQ ID NO: 367; or (iii) the VH domainhas amino acid sequence SEQ ID NO: 232 and the VL domain has amino acidsequence SEQ ID NO: 237; and, wherein the VH domain and VL domaincomprise one or more amino acid substitutions at the following residueswithin the framework regions, using the standard numbering of Kabat: 11,12 in HFW1; 37, 48 in HFW2; 68, 84, 85 in HFW3; 105, 108, 113 in HFW4;1, 2, 3, 9 in LFW1; 38, 42 in LFW2; or 58, 65, 66, 70, 74, 85, 87 inLFW3.
 31. The method according to claim 30 wherein the amino acidsubstitutions in the framework regions comprise one or moresubstitutions as shown in FIGS. 3 and
 4. 32. The method according toclaim 30 wherein the antibody molecule is an scFv.
 33. The methodaccording to claim 30 wherein the antibody molecule comprises anantibody constant region.
 34. The method according to claim 30 whereinthe antibody molecule is an IgG1, IgG2 or IgG4 molecule.